Methods for making L-glufosinate

ABSTRACT

Methods for the production of L-glufosinate (also known as phosphinothricin or (S)-2-amino-4-(hydroxy(methyl)phosphonoyl)butanoic acid) are provided. The methods comprise a two-step process. The first step involves the oxidative deamination of D-glufosinate to PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid). The second step involves the specific amination of PPO to L-glufosinate, using an amine group from one or more amine donors. By combining these two reactions, the proportion of L-glufosinate in a mixture of L-glufosinate and D-glufosinate can be substantially increased.

CROSS-REFERENCE TO PRIORITY APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/997,133, filed Aug. 19, 2020, which is a continuation of U.S.application Ser. No. 16/287,290, filed Feb. 27, 2019, which is acontinuation of U.S. application Ser. No. 15/787,448, filed Oct. 18,2017, now U.S. Pat. No. 10,260,078, which is a division of U.S.application Ser. No. 15/445,254, filed Feb. 28, 2017, now U.S. Pat. No.9,834,802, which claims priority to U.S. Provisional Application No.62/302,421, filed Mar. 2, 2016; U.S. Provisional Application No.62/336,989, filed May 16, 2016; and U.S. Provisional Application No.62/413,240, filed Oct. 26, 2016. These applications are incorporatedherein by reference in their entireties.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The Sequence Listing, which is a part of the present disclosure, issubmitted concurrently with the specification as a text file. The nameof the text file containing the Sequence Listing is“M202647D_Seqlisting.txt”, which was created on Jun. 30, 2021 and is11,437 bytes in size. The subject matter of the Sequence Listing isincorporated herein in its entirety by reference.

FIELD

Described herein are methods for producing a single stereoisomer ofglufosinate, particularly for the production of L-glufosinate.

BACKGROUND

The herbicide glufosinate is a non-selective, foliarly-applied herbicideconsidered to be one of the safest herbicides from a toxicological orenvironmental standpoint. Current commercial chemical synthesis methodsfor glufosinate yield a racemic mixture of L- and D-glufosinate (Duke etal. 2010 Toxins 2:1943-1962). However, L-glufosinate (also known asphosphinothricin or (S)-2-amino-4-(hydroxy(methyl)phosphonoyl)butanoicacid) is much more potent than D-glufosinate (Ruhland et al. (2002)Environ. Biosafety Res. 1:29-37).

Therefore, methods are needed to produce only or primarily the active,L-glufosinate form. Previously, cost effective methods to generate pureL-glufosinate, or a mixture of D- and L-glufosinate enriched forL-glufosinate, have not been available. Described herein are new andcost-effective methods for the production of L-glufosinate.

SUMMARY

Compositions and methods for making L-glufosinate are provided. Thefirst step of the process involves the oxidative deamination ofD-glufosinate to PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid).The second step involves the specific amination of PPO to L-glufosinate,using an amine group from one or more amine donors. In some embodiments,the method involves reacting D-glufosinate with a D-amino acid oxidase(DAAO) enzyme to form PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyricacid); followed by aminating the PPO to L-glufosinate by a transaminase(TA) enzyme, using an amine group from one or more amine donors, whereinat least 70% of the D-glufosinate is eliminated and/or the yield ofL-glufosinate is at least 85% of the input racemic glufosinate or atleast 70 to 85% of the D-glufosinate is converted to L-glufosinate. Insome embodiments, unreacted amine donor from one reaction can be reusedin further rounds of reaction. Optionally, the D-glufosinate isoriginally present (i.e., in the reacting step) in a racemic mixture ofD- and L-glufosinate.

The DAAO enzyme must have an increased activity of about 3 umol/min*mgor greater to drive the reaction. DAAO enzymes are available in the artand can be modified or mutated to have the necessary increased activityneeded to drive the process. In this manner, mutant or modified enzymesfrom Rhodosporidium toruloides (UniProt P80324), Trigonopsis variabilis(UniProt Q99042), Neolentinus lepideus (GenBank KZT28066.1), Trichodermareesei (GenBank XP 006968548.1), or Trichosporon oleaginosus(KLT40252.1) can be used. In some embodiments, the DAAO enzyme is amutant DAAO based on the sequence from Rhodosporidium toruloides. Whilea number of mutations can be made and tested for the effect on activity,the mutant DAAO in some embodiments may comprise mutations at positions54, 56, 58, 213, and/or 238. For example, the mutant DAAO can compriseone or more of the following mutations at position 54: N54C, N54L, N54T,or N54V. The mutant DAAO can optionally comprise the following mutationat position 56: T56M. The mutant DAAO can optionally comprise one ormore of the following mutations at position 58: F58A, F58G, F58H, F58K,F58N, F58Q, F58R, F58S, or F58T. Optionally, the mutant DAAO cancomprise the following mutation at position 213: M213S. In someembodiments, the mutant DAAO can comprise one or more of the followingcombinations of mutations: F58K and M213S; N54T and T56M; N54V and F58Q;and/or N54V, F58Q, and M213S. In each case, the enzyme needs to have anactivity of equal to or greater than about 3 umol/min*mg, greater thanabout 4 umol/min*mg, or higher. A wild type enzyme can be used in themethods of the invention as long as the enzyme has an activity level asset forth above.

The TA enzyme may be a gabT transaminase from Escherichia coli (UniProtP22256). Alternatively, the TA enzyme may be a transaminase with thesequence identified as SEQ ID NO: 1. The TA enzyme may also be selectedon the basis of sequence similarity to SEQ ID NO: 1 and/or mutated toimprove its activity in the desired reaction. Thus, sequences having80%, 85%, 90%, 95% or greater sequence identity to SEQ ID NO: 1, basedon the BLASTP method of alignment, and retain transaminase activity areencompassed by the present invention. Any DNA sequence encoding theenzyme sequence of SEQ ID NO: 1 or variants thereof are encompassedherein as well.

The reacting step and the aminating step can be performed in a singlecontainer or in separate containers. In one embodiment, all reagents aresubstantially added at the start of the reaction. Alternatively, thereagents for the reacting step and the reagents for the aminating stepare added to the single container at different times.

Also described herein is a method for selectively controlling weeds in afield containing a crop of planted seeds or crops that may optionally beresistant to glufosinate, comprising applying an effective amount of acomposition comprising L-glufosinate at an enantiomeric excess ofgreater than 90% over D-glufosinate to the field. Also described hereinis a method for selectively controlling weeds in a field, controllingweeds in non-field areas, defoliating plants or crops, and/ordesiccating crops before harvest, comprising applying an effectiveamount of a composition comprising L-glufosinate at an enantiomericexcess of greater than 90% over D-glufosinate and more than 0.01% butless than 15% PPO to the field.

The details of one or more embodiments are set forth in the drawings andthe description below. Other features, objects, and advantages will beapparent from the description and drawings, and from the claims.

Some compositions of the invention comprise D-glufosinate, PPO, andL-glufosinate. In such compositions, L-glufosinate is present at aconcentration equal to or greater than 80%, greater than 90%, greaterthan 95%, greater than 96%, greater than 97%, greater than 98%, orgreater than 99% by weight of the composition, based on the combinedweight of D-glufosinate, PPO, and L-glufosinate. Other compositionscomprise L-glufosinate at concentrations equal to 80% or greater afterisolation of the L-glufosinate from the present reaction mixture.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an exemplary conversion of D-glufosinate toL-glufosinate. The amine donor and keto acid product are examples andare not intended to be limiting.

FIG. 2 is a graph showing concentrations of L-glufosinate (circles),D-glufosinate (triangles), and PPO (squares) during a one-stepde-racemization by a N54T, T56M, F58K, and M213S mutant variant ofRhodosporidium toruloides DAAO and the E. coli gabT transaminase.

DETAILED DESCRIPTION

Compositions and methods for the production of L-glufosinate (also knownas phosphinothricin or(S)-2-amino-4-(hydroxy(methyl)phosphonoyl)butanoic acid) are provided.The methods comprise a two-step process, which may optionally occur in asingle vessel and nearly simultaneously. The first step involves theoxidative deamination of D-glufosinate to PPO(2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid). The second stepinvolves the specific amination of PPO to L-glufosinate, using an aminegroup from one or more amine donors. By combining these two reactions,the proportion of L-glufosinate can be substantially increased in aracemic glufosinate mixture. Thus, provided herein are methods to obtaina composition consisting substantially of L-glufosinate. SinceL-glufosinate is more potent than D-glufosinate, smaller amounts of thecomposition are needed to be effective as a herbicide.

In one embodiment, described herein, is a composition comprising amixture of L-glufosinate, PPO, and D-glufosinate, where L-glufosinate isthe predominant compound among the mixture of L-glufosinate, PPO, andD-glufosinate. Such composition can be used directly as a herbicide asPPO can contribute herbicidal activity (EP0030424). In otherembodiments, L-glufosinate can be purified or substantially purified andused as a herbicide.

Compositions of L-glufosinate may comprise D-glufosinate, PPO, andL-glufosinate. Optionally, the amount of L-glufosinate is 80% orgreater, 85% or greater, 90% or greater, or about 95% or greater, 97% orgreater, 98% or greater based on the combined weight of D-glufosinate,PPO, and L-glufosinate. Optionally, the amount of D-glufosinate is 10%or less, 5% or less, 2.5% or less, or 1% or less based on the combinedweight of D-glufosinate, PPO, and L-glufosinate. Optionally, the amountof PPO is more than 1% but less than 20%, less than 15%, less than 10%,or less than 5% based on the weight of D-glufosinate, PPO, andL-glufosinate. These compositions can optionally occur as dried powdersor dissolved in aqueous or non-aqueous carrier and additional chemicalspecies can optionally be present. Optionally, the composition isprepared and used in an ex vivo environment.

It is also recognized that the L-glufosinate can be further isolated andused in formulations as a herbicide.

Also described herein are formulations. The formulations compriseL-glufosinate ammonium in an amount from 10-30% by weight of theformulation; one or more additional components selected from the groupconsisting of sodium alkyl ether sulfate in an amount from 10-40% byweight of the formulation; 1-methoxy-2-propanol in an amount from 0.5-2%by weight of the formulation; dipropylene glycol in an amount from 4-18%by weight of the formulation; and alkyl polysaccharide in an amount from4-20% by weight of the formulation; and water as the balance of theformulation. Optionally, the formulation comprises L-glufosinateammonium in an amount of 12.25% by weight of the formulation; sodiumalkyl ether sulfate in an amount of 31.6% by weight of the formulation;1-methoxy-2-propanol in an amount of 1% by weight of the formulation;dipropylene glycol in an amount of 8.6% by weight of the formulation;alkyl polysaccharide in an amount of 9.8% by weight of the formulation;and water in an amount of 36.75% by weight of the formulation.Optionally, the formulation comprises L-glufosinate ammonium in anamount of 24.5% by weight of the formulation; sodium alkyl ether sulfatein an amount of 31.6% by weight of the formulation; 1-methoxy-2-propanolin an amount of 1% by weight of the formulation; dipropylene glycol inan amount of 8.6% by weight of the formulation; alkyl polysaccharide inan amount of 9.8% by weight of the formulation; and water in an amountof 24.5% by weight of the formulation. Optionally, the formulationcomprises L-glufosinate ammonium in an amount of 12.25% by weight of theformulation; sodium alkyl ether sulfate in an amount of 15.8% by weightof the formulation; 1-methoxy-2-propanol in an amount of 0.5% by weightof the formulation; dipropylene glycol in an amount of 4.3% by weight ofthe formulation; alkyl polysaccharide in an amount of 4.9% by weight ofthe formulation; and water in an amount of 62.25% by weight of theformulation. Optionally, the formulation comprises L-glufosinateammonium in an amount of 24.5% by weight of the formulation;alkylethersulfate, sodium salt in an amount of 22.1% by weight of theformulation; 1-methoxy-2-propanol in an amount of 1.0% by weight of theformulation; alkyl polysaccharide in an amount of 6.2% by weight of theformulation; and water in an amount of 46.2% by weight of theformulation.

While the methods can be used to produce a substantially purifiedL-glufosinate in a batch reaction, it is recognized that a continuousprocess can be used.

I. Methods of Synthesis

Methods for the conversion of D-glufosinate to L-glufosinate areprovided. The methods described herein provide a means for converting alow cost feedstock of a racemic mixture of D- and L-glufosinate into amore valuable product that has been enriched for L-glufosinate. Themethods for conversion includes two steps, which can occur in one ormore separate containers. The first step is the oxidative deamination ofD-glufosinate (which can be present in a racemic mixture of D- andL-glufosinate) to PPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyricacid). This step can be catalyzed by a D-amino acid oxidase (DAAO)enzyme, a D-amino acid dehydrogenase (DAAD) enzyme, or by chemicalconversion. The second step is the specific amination of PPO toL-glufosinate, using an amine group from one or more amine donors. Suchamine donors can be selected from glutamate, L-glutamate, lysine,alanine, isopropylamine, sec-butylamine, phenylethylamine and the like.This step can be catalyzed by a transaminase (TA) enzyme, an L-aminoacid dehydrogenase (LAAD) enzyme, or by chemical conversion. Using themethods described herein, compositions of substantially purifiedL-glufosinate can be obtained.

FIG. 1 sets forth an example of the conversion of D-glufosinate toL-glufosinate. As noted above, the method involves a two-step process.As illustrated, the first step is an oxidative deamination ofD-glufosinate to PPO and the second step is an amination of PPO toL-glufosinate.

The first step, i.e., the oxidative deamination of D-glufosinate to PPO,can be catalyzed by several classes of enzymes or can occurnon-enzymatically. Such enzymes include DAAO, DAAD, and D-amino aciddehydratase.

In one embodiment a DAAO enzyme is used to catalyze the conversion ofD-glufosinate to PPO. Such a reaction has the following stoichiometry:D-glufosinate+O₂+H₂O=>H₂O₂+NH₃+PPO.

Since the solubility of oxygen in aqueous reaction buffer is typicallylow compared to that of glufosinate, for an efficient process, oxygenmust be introduced throughout the time period of the DAAO reaction. Thisis in contrast to, for example, the Hawkes reaction set forth in U.S.Pat. Nos. 7,723,576; 7,939,709; 8,642,836; and 8,946,507 in which thereaction was conducted in a sealed vessel. Initially, D-glufosinate ispresent at greater than 30 g/L up to as much as 140 g/L. The initialoxygen level is typically impacted by the reaction temperature, but istypically initially present at approximately 8 mg/L and is addedthroughout the reaction to allow for sufficient oxygen for the reactionto continue apace. Water is typically, but not obligately, present atgreater than 500 g/L.

Several DAAO enzymes are known in the art and can be used in the methodsdescribed herein, as long as they are capable of accepting D-glufosinateas a substrate and provide an activity sufficient to level to drive thereaction. The DAAO enzymes useful in the methods of the invention havean activity of equal to or greater than about 3 umol/min*mg, greaterthan about 4 umol/min*mg, or higher. A wild type enzyme can be used inthe methods of the invention as long as the enzyme has an activity levelas set forth above. Such DAAO enzymes that can be used in the methodinclude those from Rhodosporidium toruloides, Trigonopsis variabilis,Fusarium sp, Candida sp, Schizosasaccharomyces sp, Verticillium sp,Neolentinus lepideus, Trichoderma reesei, Trichosporon oleaginosus, andthe like that have been modified to increase activity. Any DAAO enzymecan be used as a starting enzyme including those having sequencescorresponding to Swissprot accession numbers P80324, Q99042, P00371, andP24552 or SPTREMBL numbers Q9HGY3 and Q9Y7N4 or GenBank numbersKZT28066.1, XP_006968548.1, and KLT40252.1. The DNA sequences whichencode the DAAO may be selected from sequences set forth in EMBLaccessions A56901, RGU60066, Z50019, SSDA04, D00809, AB042032, RCDAAOX,A81420, and SPCC1450, or may be codon optimized from the proteinsequences indicated above for optimal expression in the chosenexpression host(s). U.S. Pat. No. 8,227,228 describes DAAO enzymes fromCandida intermedia. Such sequences are herein incorporated by reference.These enzymes can be modified for increased activity and used in themethods of the invention.

Additional DAAO enzymes can be identified in a variety of ways,including sequence similarity and functional screens. The DAAO enzymemay be a mutant DAAO enzyme that is capable of accepting D-glufosinateas a substrate. In Hawkes et al., supra, a mutant DAAO based on thesequence from Rhodosporidium toruloides (consisting of the F58K andM213S mutations) has been shown to accept D-glufosinate as a substrate(Hawkes et al. (2011) Plant Biotechnol J. 9(3):301-14). Other DAAOenzymes can be similarly modified to accept D-glufosinate and havegreater activity. i.e., the activity needed to drive the method of theinvention. In the same manner, known DAAO enzymes may be improved bymutagenesis, and/or novel DAAO enzymes could be identified.

In some embodiments, mutant enzymes can be made and tested in themethods described herein. Mutant DAAO enzymes (e.g., from Rhodotorulagracilis) can include one mutation, two mutations, three mutations, ormore than three mutations (e.g., four mutations, five mutations, sixmutations, seven mutations, eight mutations, nine mutations, or tenmutations or more) at positions in the mutant sequence as compared tothe wild type sequence. The mutant DAAO can optionally comprisemutations at positions 54, 56, 58, 213, and/or 238. In some embodiments,such mutants can comprise amino acid substitutions at positions 54 and56 when compared with the wild type sequence. In other embodiments, suchmutants can comprise amino acid substitutions at positions 54 and 58when compared to the wild type sequence. In other embodiments, suchmutants can include amino acid substitutions at positions 54, 213, and238 when compared with the wild type sequence.

Optionally, at position 54, the wild type asparagine may be replaced byAla, Cys, Gly, Ile, Ser, Leu, or, more preferably, Thr or Val. Forexample, the mutant DAAO can comprise one of the following mutations atposition 54: N54C, N54L, N54T, or N54V.

Optionally, at position 56, the wild type threonine can be replaced byAla, Cys, Gly, Ile, Asn, Arg, Ser, Thr, Met, or Val. See, U.S. Pat. No.7,939,709, which is incorporated herein by reference. For example, themutant DAAO can comprise the T56M mutation.

Additionally, at position 58, the wild type Phe can be replaced by Lys,Arg, Gln, Thr, Gly, Ser, Ala, Arg, Asn, or His. The mutant DAAO canoptionally comprise one of the following mutations at position 58: F58A,F58G, F58H, F58K, F58N, F58Q, F58R, F58S, or F58T. In some embodiments,the mutant DAAO does not include a mutation at position 58.

Optionally, at position 213, the wild type methionine is replaced byArg, Lys, Ser, Cys, Asn, or Ala. In some examples, the mutant DAAO cancomprise the mutation M213S.

Optionally, at position 238, the wild type tyrosine is replaced by His,Ser, Gys, Asn, or Ala.

In some embodiments, the mutant DAAO can comprise one or more of thefollowing combinations of mutations: F58K and M213S; N54T and T56M; N54Vand F58Q; N54C and F58H; N54T and F58T; N54T and F58G; N54T and F58Q;N54T and F58A; N54L and F58R; N54V and F58R; N54V and F58N; and/or N54V,F58Q, and M213S.

In one embodiment, the mutant DAAO comprises mutations in other DAAOenzymes in positions equivalent to positions 54, 56, 58, 213, and/or 238of Rhodosporidium toruloides DAAO or Trigonopsis variabilis DAAO.

Other suitable D amino acid oxidases may be obtained preferably fromfungal sources. Such DAAO enzymes can be identified and tested for usein the methods of the invention. To determine if the enzyme will acceptD-glufosinate as a substrate, an oxygen electrode assay (Hawkes, 2011,supra), colorimetric assay (Berneman A, Alves-Ferreira M, Coatnoan N,Chamond N, Minoprio P (2010) Medium/High Throughput D-Amino Acid OxidaseColorimetric Method for Determination of D-Amino Acids. Application forAmino Acid Racemases. J Microbial Biochem Technol 2: 139-146), and/ordirect measurement (via high performance liquid chromatography (HPLC),liquid chromatography mass spectrometry (LC-MS), or similar) of productformation can be employed.

The reaction catalyzed by the DAAO enzyme requires oxygen. In someembodiments, oxygen, oxygen enriched air, an oxygen enriched gas stream,or air, is introduced to the reaction, either in the head space or bysparging gas through the reaction vessel, intermittently orcontinuously, to enhance the rate of reaction. Additionally, in otherembodiments, optionally combined with sparging gas through the reactionvessel, a pressurized reactor may be used. That is, the reactor may besealed and allowed to consume O₂. Using a sealed chamber would limitvapor emissions.

When a DAAO enzyme catalyzes the conversion of D-glufosinate to PPO,hydrogen peroxide (H₂O₂) evolves. This may be damaging to enzymes andother components of the biotransformation (e.g., products and/orsubstrates). Therefore, in one embodiment, an enzyme, such as catalase,can be used in addition to the DAAO enzyme to catalyze the eliminationof hydrogen peroxide. Catalase catalyzes the decomposition of hydrogenperoxide with the following stoichiometry:2H₂O₂=>2H₂O+O₂.

In some embodiments, hydrogen peroxide can be eliminated using catalyzedand non-catalyzed decomposition reactions. For example, hydrogenperoxide can be eliminated by a non-catalyzed decomposition reactionusing increased heat and/or pH. Hydrogen peroxide can also be eliminatedby a catalyzed decomposition reaction using, for example, transitionmetals and other agents, such as potassium iodide. In addition toeliminating hydrogen peroxide, the use of catalase also produces oxygen(O₂). The production of oxygen by catalase can aid in facilitating theconversion of D-glufosinate to PPO using the DAAO enzyme, as DAAOrequires oxygen to function.

Other enzymes can be used to catalyze the conversion of D-glufosinate toPPO. For example, a DAAD enzyme that accepts D-glufosinate as asubstrate can be used with the following stoichiometry:D-glufosinate+H₂O+acceptor=>NH₃+reduced acceptor+PPO.

It is recognized that in methods where a DAAD is used, the DAADcatalyzed reaction can include redox cofactor recycling. This involvesoxidizing the reduced acceptor so that it can accept more electrons fromD-glufosinate.

In one embodiment, chemical oxidative deamination, wherein anintermediate α-keto acid is produced from the parent amino acid, can beused in the methods described herein to convert D-glufosinate toL-glufosinate. Chemical oxidative deamination involves the conversion ofan amine group to a keto group with concomitant release of ammoniatypically using metal ions such as those of copper or cobalt in anaqueous solution at temperatures between room temperature and theboiling point of the solution and at a pH in the range of about 4-about10. See, for example, Ikawa and Snell (1954) J. Am. Chem. Soc. 76 (19):4900-4902, herein incorporated by reference.

The substantially complete (greater than 70%, greater than 75%, greaterthan 80%, greater than 85%, greater than 90%, or greater than 95%)conversion of D-glufosinate to PPO can occur within 24 hours, within 18hours, within 12 hours, within 8 hours or less.

The second step of the method described herein involves the conversionof PPO to L-glufosinate using a transaminase (TA) enzyme, an L-aminoacid dehydrogenase (LAAD) enzyme, or by chemical conversion. In oneembodiment, the method is a reaction catalyzed by a TA. A TA with therequired stereospecificity that accepts PPO as a substrate catalyzes theamination of PPO to L-glufosinate with the following stoichiometry:PPO+amine donor=>L-glufosinate+keto acid.

If the reaction is conducted as a two stage process where theD-glufosinate is substantially converted to PPO in the absence of aminedonor and/or transaminase, starting amounts of PPO in the second stagetypically range from 10 g/L to 140 g/L; 20 g/L to 140 g/L; or from 30g/L to 140 g/L. If the reaction is conducted in a single stage process,the starting amounts of PPO are typically less than 1 g/L and thehighest levels of PPO during the reaction are typically less than 25g/L. The amine donor is initially present at between 1 and 50 fold molarexcess over the starting amount of racemic glufosinate.

TAs useful in the methods described herein include the gabT transaminasefrom Escherichia coli (UniProt P22256; identified herein as SEQ ID NO:3), which has been shown to catalyze the desired reaction with PPO as asubstrate (Bartsch et al. (1990) Appl Environ Microbiol. 56(1):7-12).Another enzyme has been evolved to catalyze the desired reaction at ahigher rate using isopropylamine as an amine donor (Bhatia et al. (2004)Peptide Revolution: Genomics, Proteomics & Therapeutics, Proceedings ofthe Eighteenth American Peptide Symposium, Ed. Michael Chorev and TomiK. Sawyer, Jul. 19-23, 2003, pp. 47-48). A transaminase with the aminoacid sequence of SEQ ID NO: 1 also catalyzes the desired reaction withPPO and isopropylamine as the substrate (Example 11). Additionally, TAenzymes from numerous microorganisms, such as Streptomyceshygroscopicus, Streptomyces viridochromogenes, Candida albicans, andothers can be used in the practice of the methods described herein. Inparticular, see, for example, EP0249188, and U.S. Pat. No. 5,162,212,incorporated herein by reference. Where desired, the enzymes can beevolved by mutagenesis to increase their activities. Mutant TA enzymescan be selected for desired activity by the assays outlined in Schulz etal., Appl Environ Microbiol. (1990) January 56(1):1-6, and/or by directmeasurement of the products by HPLC, LC-MS, or similar products.

Additional TA enzymes for use in the methods can be identified byscreening collections of TAs, such as those sold by Prozomix Limited(Northumberland, United Kingdom), SyncoZymes (Shanghai, China), Evocatal(Monheim am Rhein, Germany), Codexis (Redwood City, Calif.), or Abcam(Cambridge, United Kingdom) for the desired activity. Alternatively,sequence similarity can be used to identify novel TA enzymes. Finally,TA enzymes can also be identified from organisms capable of catalyzingthe desired reaction.

The selection of an appropriate amine donor is important for aneconomical conversion of D-glufosinate to L-glufosinate. A variety ofissues may be considered, including the cost of the donor, equilibriumthermodynamics, potential recovery of the donor, separation of the ketoacid product from the desired L-glufosinate, and others. Consequently,TA enzymes that accept several different amine donors can be used,including low cost amine donors such as L-aspartate or racemicaspartate, L-glutamate or racemic glutamate, L-alanine or racemicalanine, L-phenylethylamine or racemic phenylalanine, L-glycine orracemic glycine, L-lysine or racemic lysine, L-valine or racemic valine,L-serine or racemic serine, L-glutamine or racemic glutamine,isopropylamine, sec-butylamine, ethanolamine, 2-aminobutyric acid, anddiaminoproprionic acid. In some embodiments, the amine donor is notaspartate or aspartic acid (e.g., L-aspartic acid, D-aspartic acid, orracemic D,L-aspartic acid).

In embodiments where the amino donor is glutamate, the keto acidco-product that results from the transamination reaction isα-ketoglutarate (which is also referred to as α-ketoglutaric acid orα-KG). The α-ketoglutarate can be isolated and/or purified using methodsknown to those of skill in art, such as in EP Patent No. 0073711, CNPatent No. 10519873, CN Patent No. 105177065, CN Patent No. 104529755,and Zhan et al., Shipin Yu Shengwu Jishu Xuebao, 32(10): 1043-1048(2013), each of which are incorporated herein by reference in theirentireties. The produced and isolated α-ketoglutarate can be used in avariety of applications, including in synthesizing pharmaceuticalagents, food additives, and biomaterials. Optionally, theα-ketoglutarate can be chemically converted to either racemic glutamateor L-glutamate, optionally for reuse in the reaction.

Chemical reductive amination involves the conversion of a keto group toan amine group via an imine compound typically by treatment of the ketocompound with a suitable amine in an organic solution. Suitable aminesinclude, for example, methylamine or ammonia. Suitable organic solventsfor use in the organic solution include tetrahydrofuran, ethanol, ordichloromethane (DCM). The reductive amination can be performed attemperatures between room temperature and the boiling point of thesolution. The produced imine can then be reduced using a reducing agentin an organic solution. Suitable reducing agents include, for example,NaBH₄, NaHB(OAc)₃, or Na(CN)BH₃. Suitable organic solvents for use inthe organic solution include tetrahydrofuran, ethanol, or DCM. Thereduction reaction can be performed at temperatures between 0° C. andthe boiling point of the solution. Those skilled in the art will knowthat the process may be done in “one-pot” or in multiple containers,i.e., in separate transformations. Additionally, those skilled in theart will recognize that the described procedure will provide racemicamino material where possible. The use of a chiral reducing agent suchas RuCl₂[(S)-BINAP] and hydrogen gas or latent source of hydrogen gas,or achiral hydride based reducing agents in the presence chiral ligandssuch as (S)- or (R)-VAPOL in a ratio between 1:1 to 1:0.05 can produceenantiomerically pure and/or enriched amino material. See, for example,Mignonac (1921) Compt. Rend. 172:223 and G. Li, Y. Liang, J. C. Antilla(2007) J. Am. Chem. Soc., 129:5830-5831.

A wild type TA that accepts a desired amine donor can be identified, ora TA that does not normally accept a desired amine donor can be evolvedto accept the desired substrate. Optionally, the transaminase is not anaspartate transaminase. Optionally, the transaminase is not4-amino-butyrate: 2-ketoglutarate transaminase. In some embodiments, thetransaminase is not a combination enzyme system that includes aPPT-specific transaminase and glutamate:oxaloacetate transaminase.

Other enzymes for use to catalyze the conversion of PPO to L-glufosinateinclude LAAD enzymes or imine reductases that accept PPO as a substrate.Such LAAD enzymes use the following stoichiometry:NH₃+reduced acceptor+PPO=>L-glufosinate+H₂O+acceptor.

LAAD catalyzed reactions can include redox cofactor recycling, whichinvolves reducing the oxidized acceptor so that it can donate moreelectrons to PPO.

Chemical reductive amination, wherein an amino group is produced fromthe parent keto-compound, can also be used to produce glufosinate, inthe case where no chiral reductant or ligand is used, or L-glufosinate,in the case where a chiral reductant or ligand is used. Chemicalreductive amination can be affected as described above.

The substantially complete conversion of PPO to L-glufosinate may occurwithin 24 hours, within 18 hours, within 12 hours, within 8 hours, orwithin 4 hours. Substantially complete, in this context, means that theconversion of PPO to L-glufosinate is greater than about 70%, greaterthan about 75%, greater than about 80%, greater than about 85%, greaterthan about 90%, greater than about 95%, greater than about 98%, orgreater than about 99%.

If the reaction occurs in a single container or vessel, the TA enzymecan be added with the DAAO enzyme or added at a later time, e.g., afterthe DAAO enzyme has been allowed to catalyze some or substantially allof the oxidative deamination.

Enzymes can be added to the reaction by a number of methods. Oneapproach is to express the enzyme(s) in microorganism(s) such as E.coli, S. cerevisiae, P. pastoris, and others, and to add the whole cellsto the reactions as whole cell biocatalysts. Another approach is toexpress the enzyme(s), lyse the microorganisms, and add the cell lysate.Yet another approach is to purify, or partially purify, the enzyme(s)from a lysate and add pure or partially pure enzyme(s) to the reaction.If multiple enzymes are required for a reaction, the enzymes can beexpressed in one or several microorganisms, including expressing allenzymes within a single microorganism.

A further approach, which can be combined with the above approaches, isto immobilize enzyme(s) to a support (exemplary strategies are outlinedin Datta et al. (2013) 3 Biotech. February; 3(1): 1-9). As outlined inDatta et al., and not intending to be limiting, enzymes, either singlyor in combination, can, for example, be adsorbed to, or covalently ornon-covalently attached to, or entrapped within, natural or syntheticpolymers or inorganic supports, including aggregates of the enzyme(s)themselves. Once immobilized, the enzyme(s) and support can be dispersedinto bulk solution or packed into beds, columns, or any number ofsimilar approaches to interacting reaction solution with the enzymes.Since aeration is important for the DAAO reaction envisioned here,bubble columns or similar may be used for enzyme immobilization. Asexamples, reaction mixture can be flowed through a column of immobilizedenzymes (flow reaction), added to a fixed bed or column of immobilizedenzymes, allowed to react, and either removed from the bottom or top ofthe reaction vessel (plug flow), or added to dispersed immobilizedenzymes and allowed to react then the immobilized enzymes removed byfiltration, centrifugation, or similar (batch). Thus, any method forimmobilization of the enzymes may be employed in the methods of theinvention.

The DAAO, TA, and/or other reactions can occur in a buffer. Exemplarybuffers commonly used in biotransformation reactions include Tris,phosphate, or any of Good's buffers, such as2-(N-morpholino)ethanesulfonic acid (MES); N-(2-Acetamido)iminodiaceticacid (ADA); piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES);N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES);β-Hydroxy-4-morpholinepropanesulfonic acid (MOPSO); cholamine chloride;3-(N-morpholino)propanesulfonic acid (MOPS);N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid(TES); 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES);3-(Bis(2-hydroxyethyl)amino)-2-hydroxypropane-1-sulfonic acid (DIPSO);acetamidoglycine,3-(N-Tris(hydroxymethyl)methylamino(-2-hydroxypropanesulfonic acid(TAPSO); Piperazine-N, N′-bis(2-hydroxypropanesulfonic acid) (POPSO);4-(2-Hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) (HEPPSO);3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS);tricine; glycinamide; bicine; or3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonicacid (TAPS). Additional exemplary buffer recipes can be found inWhittall, J. and Sutton, P. W. (eds) (2012) Front Matter, in PracticalMethods for Biocatalysis and Biotransformations 2, John Wiley & Sons,Ltd, Chichester, UK. In some embodiments, ammonium can act as a buffer.One or more organic solvents can also be added to the reaction.

Surprisingly, the DAAO, TA, and/or other reactions can occur with no orlow levels (less than 1 mM) of buffer added (other than ammonium thatmay optionally be present due to addition of racemic glufosinateammonium). In particular, immobilized DAAO and TA may be stable andactive in the presence of less than 1 mM phosphate buffer and with noother buffer except any ammonium present due to the addition of racemicglufosinate ammonium.

The racemic glufosinate starting material can be provided in a number offorms. Various salts of racemic glufosinate, such as ammonium andhydrochloride, or the zwitterion, can be used. The racemic glufosinatemay be in the form of a solid powder (such as a powder of greater than80%, 85%, 90%, or 95% purity) or an aqueous solution (such as a roughly50% solution of racemic glufosinate).

In some embodiments, the reaction occurs within a defined pH range,which can be between pH 4 to pH 10 (e.g., between pH 6 and pH 9, such asapproximately pH 7.5 to pH 8).

In some embodiments, the reaction occurs at a defined temperature. Thetemperature can be kept at a point between room temperature and theboiling point of the solvent, most typically between room temperatureand 50° C.

As indicated, the methods described herein provide a composition ofsubstantially pure L-glufosinate (rather than a racemic mixture ofL-glufosinate and D-glufosinate). Substantially pure L-glufosinate meansthat greater than about 70%, greater than about 75%, greater than about80%, greater than about 85%, greater than about 90%, greater than about95%, greater than about 96%, greater than about 97%, greater than about98%, or greater than about 99% of the D-glufosinate has been convertedto L-glufosinate resulting in a composition having greater than about80%, greater than about 85%, greater than about 90%, greater than about95%, greater than about 96%, greater than about 97%, greater than about98%, or greater than about 99% L-glufosinate compared to the sum of theD-glufosinate and the L-glufosinate present in the composition.

In one embodiment, the L-glufosinate is not isolated from thebiotransformation mixture and a composition comprising D-glufosinate,PPO, and L-glufosinate is obtained. This composition will contain atleast 80% L-glufosinate by weight of the sum of L-glufosinate,D-glufosinate, and PPO, at least 90% L-glufosinate by weight of the sumof the components. This composition may be used directly as a herbicidalcomposition or as an ingredient in a formulated herbicidal product.

Alternatively, some or all of the components other than L-glufosinatecan be removed from the biotransformation mixture, the mixtureoptionally concentrated, and then the mixture can be used directly(and/or with the addition of various adjuvants) for the prevention orcontrol of weeds. The biotransformation mixture, in some instances, canbe used directly (and/or with the addition of various adjuvants) for theprevention or control of weeds.

Additional steps to further purify the L-glufosinate can be added. Suchfurther purification and isolation methods include ion exchange,extraction, salt formation, crystallization and filtration; each may beused multiple times or in suitable combination. Enzymes can be removedby simple filtration if supported, or if free in solution by the use ofultrafiltration, the use of absorbants like celite, cellulose or carbon,or denaturation via various techniques known to those skilled in theart.

Ion exchange processes effect separation by selective adsorption ofsolutes onto resins chosen for this purpose. Because products andimpurities must be dissolved in a single solution prior to adsorption,concentration of the purified product stream by evaporation ordistillation prior to isolation is usually required. Examples of the useof ion exchange for purification are described by Schultz et al., and inEP0249188(A2).

Purification may be achieved by the formation of an insoluble salt ofL-glufosinate by the addition of a suitable acid, including hydrochloricacid, sulfuric acid, phosphoric acid, nitric acid, acetic acid and thelike. Similarly, the purification may be achieved by the addition of asuitable base to form an insoluble salt. Useful bases includehydroxides, carbonates, sulfates and phosphates of alkali metals orhydroxides, carbonates, sulfates and phosphates of alkali earth metals.Other bases which contain nitrogen may be used, including ammonia,hydroxylamine, isopropylamine, triethylamine, tributylamine, pyridine,2-picoline, 3-picoline, 4-picoline, 2,4-lutidine, 2,6-lutidine,morpholine, N-methymorpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene, anddimethylethanolamine. It may be advantageous to concentrate the mixtureor to add a solvent (or both) to maximize yield and optimize purity ofthe desired salt. Solvents suitable for this purpose include those inwhich the solubility of the desired salt is very low (such solvents areoften called “anti-solvents”). Salts of L-glufosinate can be transformedinto forms of glufosinate suitable for formulation by standard methodsknown to those skilled in the art. Alternatively, the L-glufosinate canbe isolated as a zwitterion.

U.S. Pat. No. 9,255,115 B2 describes how the hydrochloric acid salt ofL-glufosinate can be converted to the zwitterionic form with a base suchas sodium hydroxide or sodium methoxide and then crystallized fromaqueous alcohol solvent to afford L-glufosinate in relatively highpurity. This method has the advantage of producing crystallineL-glufosinate that is not hygroscopic and therefore maintains a higherpurity compared to amorphous L-glufosinate when exposed to humidity overtime.

Other salts of L-glufosinate are known in the art. U.S. Pat. Nos.5,767,309 and 5,869,668 teach the use of chiral alkaloid bases to formdiastereomeric salts with racemic glufosinate. Purification is achievedbecause the salt of L-glufosinate precipitates from solution in muchlarger quantity than the corresponding salt of D-glufosinate. Thereforethis method could be used with the present invention to obtainL-glufosinate with high enantiomeric excess, if desired.

Optionally, purification may be achieved by first crystallizing one ormore impurities, removing the impurities by filtration and then furtherpurifying L-glufosinate from the resulting filtrate by forming a salt aspreviously described. This is advantageous if unreacted amine donor canbe partially or completely isolated and used in subsequent reactions.Similarly, unreacted PPO that is partially or completely isolated may berecycled for use in subsequent reactions.

Extraction may be used to purify the product. DE 3920570 C2 describes aprocess in which excess glutamic acid (used as the amine donor) isprecipitated by adjusting the solution pH to 3.7 to 4.2 with sulfuricacid. After filtering the glutamic acid, the filtrate pH is lowered to1-2 whereupon other impurities are extracted into a solvent. Afterextraction and concentration, ammonia is added to the aqueous solutionto a pH of 5-7 whereupon ammonium sulfate precipitates. The ammoniumsulfate is removed by filtration and the resulting filtrate isconcentrated to afford the ammonium salt of L-glufosinate.

Isolation of L-glufosinate or its salts may be desirable, for example,for the purpose of shipping solids to the location of formulation oruse. Typical industrial methods of isolation may be used, for example, afiltration, centrifugation, etc. Isolated product often requires theremoval of water, volatile impurities and solvents (if present) andtypical industrial drying equipment may be used for this purpose.Examples of such equipment include ovens, rotating drum dryers, agitateddryers, etc. In some cases, it may be advantageous to use a spray dryer.

It is not necessary to produce a solid product after purification. Thismay be advantageous if the formulation of L-glufosinate is to occur atthe same site used for L-glufosinate production. L-glufosinate and manyof its salts are readily soluble in water, and water is a convenientliquid to use for formulating products. For example, the amine donor isisolated by filtration and the resulting filtrate is concentrated bydistillation. The pH of the filtrate may be adjusted to a desirablevalue and the resulting solution may be used as is or blended withformulation ingredients. In another example, a slurry of L-glufosinateor one of its salts may be prepared as described above and isolated byfiltration. The solid could be dissolved directly on the filter byadding water or a suitable solvent to obtain a solution ofL-glufosinate.

II. Compositions

Also described herein are compositions comprising the reaction productsdescribed above. In some embodiments, the composition substantiallyincludes L-glufosinate and acceptable cationic or anionic salt formssuch as the hydrochloride, ammonium, or isopropylammonium salts. In someembodiments, the composition comprises a mixture of L-glufosinate, PPO,and D-glufosinate.

Optionally, L-glufosinate is the predominant compound amongL-glufosinate, PPO, and D-glufosinate. For example, L-glufosinate can bepresent in the composition in an amount of at least 80% by weight of thesum of L-glufosinate, PPO, and D-glufosinate, at least 85% by weight ofthe sum of L-glufosinate, PPO, and D-glufosinate, at least 90% by weightof the sum of L-glufosinate, PPO, and D-glufosinate, at least 95% byweight of the sum of L-glufosinate, PPO, and D-glufosinate, at least 96%by weight of the sum of L-glufosinate, PPO, and D-glufosinate, at least97% by weight of the sum of L-glufosinate, PPO, and D-glufosinate, atleast 98% by weight of the sum of L-glufosinate, PPO, and D-glufosinate,or at least 99% by weight of the sum of L-glufosinate, PPO, andD-glufosinate.

The composition can include PPO in an amount up to 20% by weight of thesum of L-glufosinate, PPO, and D-glufosinate. Optionally, thecomposition includes from 0.001% to 20% PPO (e.g., from 0.05% to 15% orfrom more than 0.01% to less than 5% PPO). For example, the compositioncan include PPO in an amount of less than 20%, less than 19%, less than18%, less than 17%, less than 16%, less than 15%, less than 14%, lessthan 13%, less than 12%, less than 11%, less than 10%, less than 9%,less than 8%, less than 7%, less than 6%, less than 5%, less than 4%,less than 3%, less than 2%, less than 1%, less than 0.5%, less than0.1%, or less than 0.01% by weight of the sum of the masses ofL-glufosinate, PPO, and D-glufosinate.

D-Glufosinate can be present in the composition in an amount of 15% orless by weight of the sum of L-glufosinate, PPO, and D-glufosinate. Forexample, D-glufosinate can be present in an amount of 14% or less, 13%or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less,7% or less, 8% or less, 6% or less, 5% or less, 4% or less, 3% or less,2% or less, 1% or less, or 0.5% or less by weight of the sum ofL-glufosinate, PPO, and D-glufosinate.

In some embodiments, the composition can contain small amounts (e.g.,about 10% or less, about 8% or less, about 5% or less, about 2% or less,or about 1% or less by weight of the composition) of D-glufosinate. Insome embodiments, the composition can contain small amounts (e.g., about15% or less, about 10% or less, about 8% or less, about 5% or less,about 2% or less, or about 1% or less by weight of the composition) ofPPO.

The compositions described herein are useful for application to a fieldof crop plants for the prevention or control of weeds. The compositionmay be formulated as a liquid for spraying on a field. The L-glufosinateis provided in the composition in effective amounts. As used herein,effective amount means from about 10 grams active ingredient per hectareto about 1,500 grams active ingredient per hectare, e.g., from about 50grams to about 400 grams or from about 100 grams to about 350 grams. Insome embodiments, the active ingredient is L-glufosinate. For example,the amount of L-glufosinate in the composition can be about 10 grams,about 50 grams, about 100 grams, about 150 grams, about 200 grams, about250 grams, about 300 grams, about 350 grams, about 400 grams, about 500grams, about 550 grams, about 600 grams, about 650 grams, about 700grams, about 750 grams, about 800 grams, about 850 grams, about 900grams, about 950 grams, about 1,000 grams, about 1,050 grams, about1,100 grams, about 1,150 grams, about 1,200 grams, about 1,250 grams,about 1,300 grams, about 1,350 grams, about 1,400 grams, about 1,450grams, or about 1,500 grams L-glufosinate per hectare.

The herbicidal compositions (including concentrates which requiredilution prior to application to the plants) described herein containL-glufosinate (i.e., the active ingredient), optionally some residualD-glufosinate and/or PPO, and one or more adjuvant components in liquidor solid form.

The compositions are prepared by admixing the active ingredient with oneor more adjuvants, such as diluents, extenders, carriers, surfactants,organic solvents, humectants, or conditioning agents, to provide acomposition in the form of a finely-divided particulate solid, pellet,solution, dispersion, or emulsion. Thus, the active ingredient can beused with an adjuvant, such as a finely-divided solid, a liquid oforganic origin, water, a wetting agent, a dispersing agent, anemulsifying agent, or any suitable combination of these. From theviewpoint of economy and convenience, water is the preferred diluent.However, not all the compounds are resistant to hydrolysis and in somecases this may dictate the use of non-aqueous solvent media, asunderstood by those of skill in the art.

Optionally, one or more additional components can be added to thecomposition to produce a formulated herbicidal composition. Suchformulated compositions can include L-glufosinate, carriers (e.g.,diluents and/or solvents), and other components. The formulatedcomposition includes an effective amount of L-glufosinate. Optionally,the L-glufosinate can be present in the form of L-glufosinate ammonium.The L-glufosinate ammonium can be present in an amount ranging from 10%to 30% by weight of the formulated composition. For example, theL-glufosinate ammonium can be present in an amount of 10%, 12%, 14%,16%, 18%, 20%, 22%, 24%, 26%, 28%, or 30% by weight of the formulatedcomposition. Optionally, the L-glufosinate ammonium is present in anamount of 12.25% or of 24.5%

In some examples, the formulated composition can include one or moresurfactants. A suitable surfactant for use in the formulated compositionincludes sodium alkyl ether sulfate. The surfactant can be present in anamount from 10% to 40% by weight of the formulated composition. Forexample, the surfactant can be present in an amount of 10%, 12%, 14%,16%, 18% 20% 22% 24% 26% 28% 30% 32% 34% 36% 38% or 40% by weight of theformulated composition. Optionally, the sodium alkyl ether sulfate ispresent in an amount of 11.05%, 15.8%, 22.1%, or 31.6%.

The formulated composition can optionally include one or more solvents(e.g., organic solvents). Optionally, the solvent can be1-methoxy-2-propanol, dipropylene glycol, ethylene glycol, and mixturesthereof. The one or more solvents can be present in an amount rangingfrom 0.5% to 20% by weight of the formulated composition. For example,the total amount of solvents in the composition can be present in anamount of 0.5% to 18%, 5% to 15%, or 7.5% to 10% by weight of theformulated composition.

Optionally, the solvent includes a combination of two solvents. Forexample, the solvents in the formulation can include1-methoxy-2-propanol and dipropylene glycol. The 1-methoxy-2-propanolcan be present, for example, in an amount of 0.5% to 2% by weight of theformulated composition. For example, the 1-methoxy-2-propanol can bepresent in the amount of 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1% 1.2%,1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% by weight of theformulated composition. Optionally, the 1-methoxy-2-propanol is presentin an amount of 0.5% or 1.0% by weight of the formulated composition.The dipropylene glycol can be present in an amount of from 4% to 18% byweight of the formulated composition. For example, the dipropyleneglycol can be present in an amount of 4%, 6%, 8%, 10%, 12%, 14%, 16%, or18% by weight of the formulated composition. Optionally, the dipropyleneglycol is present in an amount of 4.3% or 8.6% by weight of theformulated composition.

The formulated composition can also include one or more polysaccharidehumectants. Examples of suitable polysaccharide humectants include, forexample, alkyl polysaccharides, pentoses, high fructose corn syrup,sorbitol, and molasses. The polysaccharide humectant, such as alkylpolysaccharide, can be present in the formulated composition in anamount ranging from 4% to 20% by weight of the formulated composition.For example, the total amount of polysaccharide humectant in thecomposition can be present in an amount of 4% to 18%, 4.5% to 15%, or 5%to 10% by weight of the formulated composition. In some examples, thetotal amount of polysaccharide humectant, such as the alkylpolysaccharide, present in the formulated composition can be 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, or 18%. Optionally,the alkyl polysaccharide can be present in an amount of 3.2%, 4.9%,6.2%, or 9.8%.

A diluent can also be included in the formulated composition. Suitablediluents include water and other aqueous components. Optionally, thediluents are present in an amount necessary to produce compositionsready for packaging or for use.

In one example, the formulated composition includes L-glufosinateammonium in an amount of 12.25% by weight of the formulation; sodiumalkyl ether sulfate in an amount of 31.6% by weight of the formulation;1-methoxy-2-propanol in an amount of 1% by weight of the formulation;dipropylene glycol in an amount of 8.6% by weight of the formulation;alkyl polysaccharide in an amount of 9.8% by weight of the formulation;and water in an amount of 36.75% by weight of the formulation.

In another example, the formulated composition includes L-glufosinateammonium in an amount of 24.5% by weight of the formulation; sodiumalkyl ether sulfate in an amount of 31.6% by weight of the formulation;1-methoxy-2-propanol in an amount of 1% by weight of the formulation;dipropylene glycol in an amount of 8.6% by weight of the formulation;alkyl polysaccharide in an amount of 9.8% by weight of the formulation;and water in an amount of 36.75% by weight of the formulation.

In another example, the formulated composition includes L-glufosinateammonium in an amount of 12.25% by weight of the formulation; sodiumalkyl ether sulfate in an amount of 15.8% by weight of the formulation;1-methoxy-2-propanol in an amount of 0.5% by weight of the formulation;dipropylene glycol in an amount of 4.3% by weight of the formulation;alkyl polysaccharide in an amount of 4.9% by weight of the formulation;and water in an amount of 62.25% by weight of the formulation.

In another example, the formulated composition includes L-glufosinateammonium in an amount of 24.5% by weight of the formulation; sodiumalkyl ether sulfate in an amount of 22.1% by weight of the formulation;1-methoxy-2-propanol in an amount of 1% by weight of the formulation;alkyl polysaccharide in an amount of 6.2% by weight of the formulation;and water in an amount of 46.2% by weight of the formulation.

In another example, the formulated composition includes L-glufosinateammonium in an amount of 12.25% by weight of the formulation; sodiumalkyl ether sulfate in an amount of 22.1% by weight of the formulation;1-methoxy-2-propanol in an amount of 1% by weight of the formulation;alkyl polysaccharide in an amount of 6.2% by weight of the formulation;and water in an amount of 58.45% by weight of the formulation.

In another example, the formulated composition includes L-glufosinateammonium in an amount of 12.25% by weight of the formulation; sodiumalkyl ether sulfate in an amount of 11.05% by weight of the formulation;1-methoxy-2-propanol in an amount of 0.5% by weight of the formulation;alkyl polysaccharide in an amount of 3.1% by weight of the formulation;and water in an amount of 73.1% by weight of the formulation.

Further components suitable for use in the formulated compositionsprovided herein are described in U.S. Pat. Nos. 4,692,181 and 5,258,358,both of which are incorporated by reference herein in their entireties.

The herbicidal compositions described herein, particularly liquids andsoluble powders, can contain as further adjuvant components one or moresurface-active agents in amounts sufficient to render a givencomposition readily dispersible in water or in oil. The incorporation ofa surface-active agent into the compositions greatly enhances theirefficacy. Surface-active agent, as used herein, includes wetting agents,dispersing agents, suspending agents, and emulsifying agents areincluded therein. Anionic, cationic, and non-ionic agents can be usedwith equal facility.

Suitable wetting agents include alkyl benzene and alkyl naphthalenesulfonates, sulfated fatty alcohols, amines or acid amides, long chainacid esters of sodium isothionate, esters of sodium sulfosuccinate,sulfated or sulfonated fatty acid esters petroleum solfonates,sulfonated vegetable oils, ditertiary acetylenic glycols,polyoxyethylene derivatives of alkylphenols (particularly isooctylphenoland nonylphenol), and polyoxethylene derivatives of the mono-higherfatty acid esters of hexitol anhydrides (e.g. sorbitan). Exemplarydispersants include methyl cellulose, polyvinyl alcohol, sodium ligninsulfonates, polymeric alkyl naphthalene sulfonates, sodium naphthalenesulfonate, polymethylene bisnaphthalenesulfonate, and sodium N-methyl-N-(long chain acid) laurates.

Water-dispersible powder compositions can be made containing one or moreactive ingredients, an inert solid extender, and one or more wetting anddispersing agents. The inert solid extenders are usually of mineralorigin, such as the natural clays, diatomaceous earth, and syntheticminerals derived from silica and the like. Examples of such extendersinclude kaolinites, attapulgite clay, and synthetic magnesium silicate.Water-dispersible powders described herein can optionally contain fromabout 5 to about 95 parts by weight of active ingredient (e.g., fromabout 15 to 30 parts by weight of active ingredient), from about 0.25 to25 parts by weight of wetting agent, from about 0.25 to 25 parts byweight of dispersant, and from 4.5 to about 94.5 parts by weight ofinert solid extender, all parts being by weight of the totalcomposition. Where required, from about 0.1 to 2.0 parts by weight ofthe solid inert extender can be replaced by a corrosion inhibitor oranti-foaming agent or both.

Aqueous suspensions can be prepared by dissolution or by mixing togetherand grinding an aqueous slurry of a water-insoluble active ingredient inthe presence of a dispersing agent to obtain a concentrated slurry ofvery finely-divided particles. The resulting concentrated aqueoussuspension is characterized by its extremely small particle size, sothat when diluted and sprayed, coverage is very uniform.

Emulsifiable oils are usually solutions of active ingredient inwater-immiscible or partially water-immiscible solvents together with asurface active agent. Suitable solvents for the active ingredientdescribed herein include hydrocarbons and water-immiscible ethers,esters, or ketones. The emulsifiable oil compositions generally containfrom about 5 to 95 parts active ingredient, about 1 to 50 parts surfaceactive agent, and about 4 to 94 parts solvent, all parts being by weightbased on the total weight of emulsifiable oil.

Compositions described herein can also contain other additaments, forexample, fertilizers, phytotoxicants and plant growth regulants,pesticides, and the like used as adjuvants or in combination with any ofthe above-described adjuvants. The compositions described herein canalso be admixed with the other materials, e.g., fertilizers, otherphytotoxicants, etc., and applied in a single application.

In each of the formulation types described herein, e.g., liquid andsolid formulations, the concentration of the active ingredients are thesame.

In some embodiments, the composition can include α-ketoglutarate as themajor component. α-ketoglutarate is an important dicarboxylic acid andone of the key intermediates in the tricarboxylic acid cycle and aminoacid metabolism. α-ketoglutarate can be isolated from the reactionmixture by methods such as that set forth in French Patent No. 07199,herein incorporated by reference. The α-ketoglutarate composition can beformulated with pharmaceutical excipients and carriers, food additives,or components used to form biomaterials. The α-ketoglutarate compositioncan be used in a variety of applications, including in synthesizingpharmaceutical agents, food additives, and biomaterials, as described inLi et al., Bioprocess Biosyst Eng, 39:967-976 (2016).

It is recognized that the herbicidal compositions can be used incombination with other herbicides. The herbicidal compositions of thepresent invention are often applied in conjunction with one or moreother herbicides to control a wider variety of undesirable vegetation.When used in conjunction with other herbicides, the presently claimedcompounds can be formulated with the other herbicide or herbicides, tankmixed with the other herbicide or herbicides or applied sequentiallywith the other herbicide or herbicides. Some of the herbicides that canbe employed in conjunction with the compounds of the present inventioninclude: amide herbicides such as allidochlor, beflubutamid, benzadox,benzipram, bromobutide, cafenstrole, CDEA, chlorthiamid, cyprazole,dimethenamid, dimethenamid-P, diphenamid, epronaz, etnipromid,fentrazamide, flupoxam, fomesafen, halosafen, isocarbamid, isoxaben,napropamide, naptalam, pethoxamid, propyzamide, quinonamid and tebutam;anilide herbicides such as chloranocryl, cisanilide, clomeprop,cypromid, diflufenican, etobenzanid, fenasulam, flufenacet, flufenican,mefenacet, mefluidide, metamifop, monalide, naproanilide, pentanochlor,picolinafen and propanil; arylalanine herbicides such as benzoylprop,flamprop and flamprop-M; chloroacetanilide herbicides such asacetochlor, alachlor, butachlor, butenachlor, delachlor, diethatyl,dimethachlor, metazachlor, metolachlor, S-metolachlor, pretilachlor,propachlor, propisochlor, prynachlor, terbuchlor, thenylchlor andxylachlor; sulfonanilide herbicides such as benzofluor, perfluidone,pyrimisulfan and profluazol; sulfonamide herbicides such as asulam,carbasulam, fenasulam and oryzalin; antibiotic herbicides such asbilanafos; benzoic acid herbicides such as chloramben, dicamba,2,3,6-TBA and tricamba; pyrimidinyloxybenzoic acid herbicides such asbispyribac and pyriminobac; pyrimidinylthiobenzoic acid herbicides suchas pyrithiobac; phthalic acid herbicides such as chlorthal; picolinicacid herbicides such as aminopyralid, clopyralid and picloram;quinolinecarboxylic acid herbicides such as quinclorac and quinmerac;arsenical herbicides such as cacodylic acid, CMA, DSMA, hexaflurate,MAA, MAMA, MSMA, potassium arsenite and sodium arsenite;benzoylcyclohexanedione herbicides such as mesotrione, sulcotrione,tefuryltrione and tembotrione; benzofuranyl alkylsulfonate herbicidessuch as benfuresate and ethofumesate; carbamate herbicides such asasulam, carboxazole chlorprocarb, dichlormate, fenasulam, karbutilateand terbucarb; carbanilate herbicides such as barban, BCPC, carbasulam,carbetamide, CEPC, chlorbufam, chlorpropham, CPPC, desmedipham,phenisopham, phenmedipham, phenmedipham-ethyl, propham and swep;cyclohexene oxime herbicides such as alloxydim, butroxydim, clethodim,cloproxydim, cycloxydim, profoxydim, sethoxydim, tepraloxydim andtralkoxydim; cyclopropylisoxazole herbicides such as isoxachlortole andisoxaflutole; dicarboximide herbicides such as benzfendizone,cinidon-ethyl, flumezin, flumiclorac, flumioxazin and flumipropyn;dinitroaniline herbicides such as benfluralin, butralin, dinitramine,ethalfluralin, fluchloralin, isopropalin, methalpropalin, nitralin,oryzalin, pendimethalin, prodiamine, profluralin and trifluralin;dinitrophenol herbicides such as dinofenate, dinoprop, dinosam, dinoseb,dinoterb, DNOC, etinofen and medinoterb; diphenyl ether herbicides suchas ethoxyfen; nitrophenyl ether herbicides such as acifluorfen,aclonifen, bifenox, chlomethoxyfen, chlomitrofen, etnipromid,fluorodifen, fluoroglycofen, fluoronitrofen, fomesafen, furyloxyfen,halosafen, lactofen, nitrofen, nitrofluorfen and oxyfluorfen;dithiocarbamate herbicides such as dazomet and metam; halogenatedaliphatic herbicides such as alorac, chloropon, dalapon, flupropanate,hexachloroacetone, iodomethane, methyl bromide, monochloroacetic acid,SMA and TCA; imidazolinone herbicides such as imazamethabenz, imazamox,imazapic, imazapyr, imazaquin and imazethapyr; inorganic herbicides suchas ammonium sulfamate, borax, calcium chlorate, copper sulfate, ferroussulfate, potassium azide, potassium cyanate, sodium azide, sodiumchlorate and sulfuric acid; nitrile herbicides such as bromobonil,bromoxynil, chloroxynil, dichlobenil, iodobonil, ioxynil and pyraclonil;organophosphorus herbicides such as amiprofos-methyl, anilofos,bensulide, bilanafos, butamifos, 2,4-DEP, DMPA, EBEP, fosamine,glyphosate and piperophos; phenoxy herbicides such as bromofenoxim,clomeprop, 2,4-DEB, 2,4-DEP, difenopenten, disul, erbon, etnipromid,fenteracol and trifopsime; phenoxyacetic herbicides such as 4-CPA,2,4-D, 3,4-DA, MCPA, MCPA-thioethyl and 2,4,5-T; phenoxybutyricherbicides such as 4-CPB, 2,4-DB, 3,4-DB, MCPB and 2,4,5-TB;phenoxypropionic herbicides such as cloprop, 4-CPP, dichlorprop,dichlorprop-P, 3,4-DP, fenoprop, mecoprop and mecoprop-P;aryloxyphenoxypropionic herbicides such as chlorazifop, clodinafop,clofop, cyhalofop, diclofop, fenoxaprop, fenoxaprop-P, fenthiaprop,fluazifop, fluazifop-P, haloxyfop, haloxyfop-P, isoxapyrifop, metamifop,propaquizafop, quizalofop, quizalofop-P and trifop; phenylenediamineherbicides such as dinitramine and prodiamine; pyrazolyl herbicides suchas benzofenap, pyrazolynate, pyrasulfotole, pyrazoxyfen, pyroxasulfoneand topramezone; pyrazolylphenyl herbicides such as fluazolate andpyraflufen; pyridazine herbicides such as credazine, pyridafol andpyridate; pyridazinone herbicides such as brompyrazon, chloridazon,dimidazon, flufenpyr, metflurazon, norflurazon, oxapyrazon and pydanon;pyridine herbicides such as aminopyralid, cliodinate, clopyralid,dithiopyr, fluroxypyr, haloxydine, picloram, picolinafen, pyriclor,thiazopyr and triclopyr; pyrimidinediamine herbicides such as iprymidamand tioclorim; quaternary ammonium herbicides such as cyperquat,diethamquat, difenzoquat, diquat, morfamquat and paraquat; thiocarbamateherbicides such as butylate, cycloate, di-allate, EPTC, esprocarb,ethiolate, isopolinate, methiobencarb, molinate, orbencarb, pebulate,prosulfocarb, pyributicarb, sulfallate, thiobencarb, tiocarbazil,tri-allate and vemolate; thiocarbonate herbicides such as dimexano, EXDand proxan; thiourea herbicides such as methiuron; triazine herbicidessuch as dipropetryn, triaziflam and trihydroxytriazine; chlorotriazineherbicides such as atrazine, chlorazine, cyanazine, cyprazine,eglinazine, ipazine, mesoprazine, procyazine, proglinazine, propazine,sebuthylazine, simazine, terbuthylazine and trietazine; methoxytriazineherbicides such as atraton, methometon, prometon, secbumeton, simetonand terbumeton; methylthiotriazine herbicides such as ametryn,aziprotryne, cyanatryn, desmetryn, dimethametryn, methoprotryne,prometryn, simetryn and terbutryn; triazinone herbicides such asametridione, amibuzin, hexazinone, isomethiozin, metamitron andmetribuzin; triazole herbicides such as amitrole, cafenstrole, epronazand flupoxam; triazolone herbicides such as amicarbazone, bencarbazone,carfentrazone, flucarbazone, propoxycarbazone, sulfentrazone andthiencarbazone-methyl; triazolopyrimidine herbicides such ascloransulam, diclosulam, florasulam, flumetsulam, metosulam, penoxsulamand pyroxsulam; uracil herbicides such as butafenacil, bromacil,flupropacil, isocil, lenacil and terbacil; 3-phenyluracils; ureaherbicides such as benzthiazuron, cumyluron, cycluron, dichloralurea,diflufenzopyr, isonoruron, isouron, methabenzthiazuron, monisouron andnoruron; phenylurea herbicides such as anisuron, buturon, chlorbromuron,chloreturon, chlorotoluron, chloroxuron, daimuron, difenoxuron,dimefuron, diuron, fenuron, fluometuron, fluothiuron, isoproturon,linuron, methiuron, methyldymron, metobenzuron, metobromuron, metoxuron,monolinuron, monuron, neburon, parafluron, phenobenzuron, siduron,tetrafluron and thidiazuron; pyrimidinylsulfonylurea herbicides such asamidosulfuron, azimsulfuron, bensulfuron, chlorimuron, cyclosulfamuron,ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron,foramsulfuron, halosulfuron, imazosulfuron, mesosulfuron, nicosulfuron,orthosulfamuron, oxasulfuron, primisulfuron, pyrazosulfuron,rimsulfuron, sulfometuron, sulfosulfuron and trifloxysulfuron;triazinylsulfonylurea herbicides such as chlorsulfuron, cinosulfuron,ethametsulfuron, iodosulfuron, metsulfuron, prosulfuron, thifensulfuron,triasulfuron, tribenuron, triflusulfuron and tritosulfuron;thiadiazolylurea herbicides such as buthiuron, ethidimuron, tebuthiuron,thiazafluron and thidiazuron; and unclassified herbicides such asacrolein, allyl alcohol, aminocyclopyrachlor, azafenidin, benazolin,bentazone, benzobicyclon, buthidazole, calcium cyanamide, cambendichlor,chlorfenac, chlorfenprop, chlorflurazole, chlorflurenol, cinmethylin,clomazone, CPMF, cresol, ortho-dichlorobenzene, dimepiperate, endothal,fluoromidine, fluridone, flurochloridone, flurtamone, fluthiacet,indanofan, methazole, methyl isothiocyanate, nipyraclofen, OCH,oxadiargyl, oxadiazon, oxaziclomefone, pentachlorophenol, pentoxazone,phenylmercury acetate, pinoxaden, prosulfalin, pyribenzoxim, pyriftalid,quinoclamine, rhodethanil, sulglycapin, thidiazimin, tridiphane,trimeturon, tripropindan and tritac. The herbicidal compositions of thepresent invention can, further, be used in conjunction with glyphosateor 2,4-D on glyphosate-tolerant or 2,4-D-tolerant crops. It is generallypreferred to use the compositions of the invention in combination withherbicides that are selective for the crop being treated and whichcomplement the spectrum of weeds controlled by these compositions at theapplication rate employed. It is further generally preferred to applythe compositions of the invention and other complementary herbicides atthe same time, either as a combination formulation or as a tank mix.

III. Methods of Use of L-Glufosinate Compositions

The compositions described herein can be used in methods for selectivelycontrolling weeds in a field or any other area, including, for example,a railway, lawn, golf course, and others where the control of weeds isdesired. Optionally, the field or other area can contain a crop ofplanted seeds or crops that are resistant to glufosinate. The methodscan include applying an effective amount of a composition comprisingL-glufosinate as described herein to the field.

The compositions described herein are useful for application to a fieldof crop plants for the prevention or control of weeds. The compositionmay be formulated as a liquid for spraying on a field. The L-glufosinateis provided in the composition in effective amounts. As used herein,effective amount means from about 10 grams active ingredient per hectareto about 1,500 grams active ingredient per hectare, e.g., from about 50grams to about 400 grams or from about 100 grams to about 350 grams. Insome embodiments, the active ingredient is L-glufosinate. For example,the amount of L-glufosinate in the composition can be about 10 grams,about 50 grams, about 100 grams, about 150 grams, about 200 grams, about250 grams, about 300 grams, about 350 grams, about 400 grams, about 500grams, about 550 grams, about 600 grams, about 650 grams, about 700grams, about 750 grams, about 800 grams, about 850 grams, about 900grams, about 950 grams, about 1,000 grams, about 1,050 grams, about1,100 grams, about 1,150 grams, about 1,200 grams, about 1,250 grams,about 1,300 grams, about 1,350 grams, about 1,400 grams, about 1,450grams, or about 1,500 grams L-glufosinate per hectare.

IV. Exemplary Embodiments

Non-limiting embodiments include:

1. A method for making L-glufosinate, comprising:

reacting D-glufosinate with a D-amino acid oxidase (DAAO) enzyme to formPPO (2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid); and

aminating the PPO to L-glufosinate by a transaminase (TA) enzyme, usingan amine group from one or more amine donors,

wherein at least 70% of the D-glufosinate is converted to L-glufosinate.

2. The method of embodiment 1, wherein the amine donor is selected fromthe group consisting of glutamate, L-glutamate, alanine, sec-butylamine,phenylethylamine, glycine, lysine, valine, serine, glutamine,isopropylamine, ethanolamine, 2-aminobutyric acid, and diaminoproprionicacid, or any secondary amine or amino acid.

3. The method of embodiment 1, wherein the D-glufosinate is originallypresent in a racemic mixture of D- and L-glufosinate or salts thereof.

4. The method of embodiment 1, wherein the DAAO enzyme is selected fromthe enzyme from Rhodosporidium toruloides or Trigonopsis variabilis,Neolentinus lepideus, Trichoderma reesei, or Trichosporon oleaginosus.In an embodiment, the Rhodosporidium toruloides DAAO enzyme is UniProtP80324. In an embodiment, the Trigonopsis variabilis DAAO enzyme isUniProt Q99042. In an embodiment, the Neolentinus lepideus DAAO enzymeis KZT28066.1. In an embodiment, the Trichoderma reesei DAAO enzyme isXP_006968548.1. In an embodiment, the Trichosporon oleaginosus DAAOenzyme is KLT40252.1.

5. The method of embodiment 1, wherein the DAAO enzyme is a mutant DAAO.

6. The method of embodiment 5, wherein the mutant DAAO is a mutant DAAObased on the sequence from Rhodosporidium toruloides.

7. The method of embodiment claim 5, wherein the mutant DAAO comprisesone or more mutations at positions 54, 56, 58, 213, and 238.

8. The method of embodiment 7, wherein the mutation at position 54 isselected from the group consisting of N54C, N54L, N54T, and N54V.

9. The method of embodiment 7, wherein the mutation at position 56 isT56M.

10. The method of embodiment 7, wherein the mutation at position 58 isselected from the group consisting of F58A, F58G, F58H, F58K, F58N,F58Q, F58R, F58S, and F58T.

11. The method of embodiment 7, wherein the mutation at position 213 isM213S.

12. The method of embodiment 5, wherein the mutant DAAO comprisesmutations F58K and M213S.

13. The method of embodiment 5, wherein the mutant DAAO comprisesmutations at positions 54 and 56.

14. The method of embodiment 5, wherein the mutant DAAO comprisesmutations N54T and T56M.

15. The method of embodiment 5, wherein the mutant DAAO comprisesmutations F58Q or F58H.

16. The method of embodiment 5, wherein the mutant DAAO comprisesmutations N54V and F58Q.

17. The method of embodiment 5, wherein the mutant DAAO comprisesmutations N54V, F58Q, and M213S.

18. The method of embodiment 1, wherein the TA enzyme is a gabTtransaminase from Escherichia coli. In an embodiment, the Escherichiacoli gabT transaminase is UniProt P22256.

19. The method of embodiment 1, wherein the TA enzyme is encoded by SEQID NO: 1.

20. The method of embodiment 1, wherein the reacting step and theaminating step are performed in a single container.

21. The method of embodiment 20, wherein all reagents are substantiallyadded at the start of the reaction.

22. The method of embodiment 20, wherein the reagents for the reactingstep and the reagents for the aminating step are added to the singlecontainer at different times.

23. The method of embodiment 1, wherein the reacting step and theaminating step are performed in separate containers.

24. A composition comprising D-glufosinate, PPO, and L-glufosinate.

25. The composition of embodiment 24, wherein the amount ofL-glufosinate is 90% or greater based on the total amount ofD-glufosinate, PPO, and L-glufosinate.

26. The method of embodiment 1 wherein a solid is obtained having thecomposition of embodiment 24 or 25.

27. The method of embodiment 1 wherein a solution of L-glufosinate isobtained for use in a formulation which has herbicidal activity.

28. A formulation, comprising L-glufosinate ammonium in an amount from10-30% by weight of the formulation; one or more additional componentsselected from the group consisting of sodium alkyl ether sulfate in anamount from 10-40% by weight of the formulation; 1-methoxy-2-propanol inan amount from 0.5-2% by weight of the formulation; dipropylene glycolin an amount from 4-18% by weight of the formulation; and alkylpolysaccharide in an amount from 4-20% by weight of the formulation; andwater as the balance of the formulation.

29. The composition of embodiment 28, wherein the formulation comprises:L-glufosinate ammonium in an amount of 12.25% by weight of theformulation; sodium alkyl ether sulfate in an amount of 31.6% by weightof the formulation; 1-methoxy-2-propanol in an amount of 1% by weight ofthe formulation; dipropylene glycol in an amount of 8.6% by weight ofthe formulation; alkyl polysaccharide in an amount of 9.8% by weight ofthe formulation; and water in an amount of 36.75% by weight of theformulation.

30. The composition of embodiment 28, wherein the formulation comprises:L-glufosinate ammonium in an amount of 24.5% by weight of theformulation; sodium alkyl ether sulfate in an amount of 31.6% by weightof the formulation; 1-methoxy-2-propanol in an amount of 1% by weight ofthe formulation; dipropylene glycol in an amount of 8.6% by weight ofthe formulation; alkyl polysaccharide in an amount of 9.8% by weight ofthe formulation; and water in an amount of 24.5% by weight of theformulation.

31. The composition of embodiment 28, wherein the formulation comprises:L-glufosinate ammonium in an amount of 12.25% by weight of theformulation; sodium alkyl ether sulfate in an amount of 15.8% by weightof the formulation; 1-methoxy-2-propanol in an amount of 0.5% by weightof the formulation; dipropylene glycol in an amount of 4.3% by weight ofthe formulation; alkyl polysaccharide in an amount of 4.9% by weight ofthe formulation; and water in an amount of 62.25% by weight of theformulation.

32. A formulation, comprising L-glufosinate ammonium in an amount from10-30% by weight of the formulation; one or more additional componentsselected from the group consisting of sodium alkyl ether sulfate in anamount from 10-40% by weight of the formulation; 1-methoxy-2-propanol inan amount from 0.5-2% by weight of the formulation; and alkylpolysaccharide in an amount from 3-10% by weight of the formulation; andwater as the balance of the formulation.

33. The composition of embodiment 32, wherein the formulation comprises:L-glufosinate ammonium in an amount of 12.25% by weight of theformulation; sodium alkyl ether sulfate in an amount of 22.1% by weightof the formulation; 1-methoxy-2-propanol in an amount of 1% by weight ofthe formulation; alkyl polysaccharide in an amount of 6.2% by weight ofthe formulation; and water in an amount of 58.45% by weight of theformulation.

34. The composition of embodiment 32, wherein the formulation comprises:L-glufosinate ammonium in an amount of 24.5% by weight of theformulation; sodium alkyl ether sulfate in an amount of 22.1% by weightof the formulation; 1-methoxy-2-propanol in an amount of 1% by weight ofthe formulation; alkyl polysaccharide in an amount of 6.2% by weight ofthe formulation; and water in an amount of 46.2% by weight of theformulation.

35. The composition of embodiment 32, wherein the formulation comprises:L-glufosinate ammonium in an amount of 12.25% by weight of theformulation; sodium alkyl ether sulfate in an amount of 11.05% by weightof the formulation; 1-methoxy-2-propanol in an amount of 0.5% by weightof the formulation; alkyl polysaccharide in an amount of 3.1% by weightof the formulation; and water in an amount of 73.1% by weight of theformulation.

36. A method for selectively controlling weeds in an area comprising:

applying an effective amount of a composition comprising L-glufosinateat an enantiomeric excess of greater than 90% over D-glufosinate to thearea.

37. The method of embodiment 36, wherein the amount of the compositionis applied at less than 400 grams of the sum of L-glufosinate andD-glufosinate per hectare.

38. A method for selectively controlling weeds in an area comprising:

applying an effective amount of a composition comprising L-glufosinateat an enantiomeric excess of greater than 90% over D-glufosinate to thearea and more than 0.01% but less than 10% PPO to the area.

39. The method of embodiment 38, wherein the amount of the compositionis applied at less than 400 grams of the sum of L-glufosinate,D-glufosinate and PPO per hectare.

40. A method for selectively controlling weeds in an area containing acrop of planted seeds or crops that are resistant to glufosinate,comprising:

applying an effective amount of a composition comprising L-glufosinateat an enantiomeric excess of greater than 90% over D-glufosinate andmore than 0.01% but less than 10% PPO to the field.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1: DAAO Enzyme Purification

The coding sequence of a mutant DAAO from Rhodosporidium toruloides (forexample, consisting of a MMARIRL leader sequence and the F58K and M213Smutations) was cloned into the pET14b vector to allow for expression ofan N terminally 6×His tagged protein. This pET14b-RgDAAO plasmid wastransformed into BL21 (BE3) trxB pLysS cells. The sequence of the wildtype DAAO from Rhodosporidium toruloides to which all numberingdescribed here corresponds, is:

(SEQ ID NO: 2) MHSQKRVVVLGSGVIGLSSALILARKGYSVHILARDLPEDVSSQTFASPWAGANWTPFMTLTDGPRQAKWEESTFKKWVELVPTGHAMWLKGTRRFAQNEDGLLGHWYKDITPNYRPLPSSECPPGAIGVTYDTLSVHAPKYCQYLARELQKLGATFERRTVTSLEQAFDGADLVVNATGLGAKSIAGIDDQAAEPIRGQTVLVKSPCKRCTMDSSDPASPAYIIPRPGGEVICGGTYGVGDWDLSVNPETVQRILKHCLRLDPTISSDGTIEGIEVLRHNVGLRPARRGGPRVEAERIVLPLDRTKSPLSLGRGSARAAKEKEVTLVHAYGFSSAGYQQSWGAAEDVAQLVDEAFQRYHGAARESKL.

To purify DAAO enzyme, cells were grown in 400 mL autoinducing medium(LB broth base with trace elements, Formedium) at 30° C. for 20 to 24hours. Cells were harvested in precooled centrifuges and buckets, washedwith cold water, centrifuged again, and stored at −80° C. untilpurification.

Cell pellets were then thawed in lysis buffer (50 mM potassiumphosphate, pH 8.0, 20 mM imidazole and 1% Sigma protease inhibitorcocktail (PIC) w/o EDTA) at a volume of 5 mL of lysis buffer per 1 gcell pellet. While on ice, cells were sonicated 4 times for 30 secondsat an amplitude of 10. The cell lysate was clarified by centrifugationand then added to cobalt resin (HisPur Cobalt, ThermoScientific) at 4times the bed volume. The cell lysate was incubated for 1 hour, withgentle shaking, at room temperature. The resin was added to a column andwashed twice with 5 bed volumes of wash buffer (50 mM Kpi, pH 8.0, 20 mMimidazole). The elution was performed 4 times with 1 bed volume ofelution buffer (50 mM Kpi, 200 mM imidazole).

Example 2: Colorimetric Determination of DAAO Activity

DAAO activity was determined similarly to Berneman et al. In brief, 100uL of substrate and HRP (0.1 mg/mL HRP, Sigma P8375, and the desiredamount of D-glufosinate or racemic D/L-glufosinate in 50 mM potassiumphosphate, pH 8) was added to a Brand UV micro cuvette. To that, 50 uLof dyes (60 ug/mL TBHBA, Sigma 439533, and 1 mg/mL 4-aminoantipyrine,Sigma A4382, in 50 mM potassium phosphate, pH 8) was added and then 50uL of enzyme mix (DAAO concentration as desired in 100 mM potassiumphosphate, pH 8). The reaction was monitored on a spectrophotometer at510 nm over an appropriate time to determine the enzyme kinetics.Although no flavin adenine dinucleotide (FAD) was added to thepurification of DAAO or the reaction, this reagent can optionally beincluded. Two exemplary mutant variants of Rhodosporidium toruloidesDAAO, AC201 (containing F58K and M213S) and AC263 (containing N54T,T56M, F58K, and M213S), purified as in Example 1, were tested using thisassay and shown to produce hydrogen peroxide, demonstrating theiractivity in oxidizing D-glufosinate. AC201 and AC263 have similarV_(max), but AC263 has a lower K_(M).

Example 3: Purification of Transaminases

To purify, for example, the Escherichia coli gabT transaminase (UniProtP22256), the gene was amplified from E. coli K12 strain ER2925 andcloned into pET-14b to generate an N-terminal 6×His tagged version. Thisplasmid was then transformed into BL21 (DE3) cells for induction. Afterinduction in autoinducing media, cells were lysed by sonication and the6×His tagged enzyme purified as described in Example 1.

Example 4: Demonstration of Transaminase Activity

In a non-limiting example, the source of PPO for a transamination assaycan be D-glufosinate or racemic D/L-glufosinate that has been convertedby a DAAO to PPO. In the first step, 39 mM racemic D/L-glufosinate wasincubated with 0.5 mg/ml purified Rhodosporidium toruloides DAAO F58KM213S and 10 ug/mL catalase in 50 mM Potassium phosphate buffer, pH8 for20 hours at 30 C. This resulted in conversion of the majority of theD-glufosinate to PPO. Subsequently, purified E. coli gabT was added at20 ug/mL and L-glutamate was added at 50 mM as the amine donor. Atrelevant points, samples were stopped by boiling for 10 minutes followedby precipitation with an equal volume of acetonitrile. The individualchemical species were resolved on an HPLC with a Chirobiotic T2 columnand quantified by comparison to authentic standards.

The combination of a mutant variant of DAAO and a transaminase resultedin an improvement of an enantiomeric enrichment that started at 0% forL-glufosinate over D-glufosinate (i.e., equal representation ofD-glufosinate and L-glufosinate) to an enantiomeric enrichment of 92%.These results demonstrate that E. coli gabT has transaminase activity,and this assay can be used to determine the activity of any number ofwild type and/or mutant potential transaminases.

Example 5: De-Racemization of Racemic D/L-Glufosinate in a Single Vessel

Reactions were set up similarly as in Example 4. The system (5.45 mL,30° C.) was run in phosphate buffer at a pH of 7.3. It was noted that 50mM of phosphate buffer at a pH of 8.0 was inadequate to buffer the aminoacid additions and that the unadjusted pH after amino acid additions inthis system was pH 6.4. The pH was adjusted using 1M of the base saltK2HPO4 to a volume of 5.45 mL, meaning that the actual initial substrateconcentration by addition was 275 mM. The following reagents were addedessentially simultaneously at the start of the reaction: 271 mgD,L-glufosinate, 420 mg glutamate, 15 mg AC263 DAAO, 50 μg catalase, and1.0 mg E. coli gab T transaminase. FIG. 2 shows that, when all reagentswere added, the amount of D-PPT (D-glufosinate) diminished with onlymodest accumulation of PPO. This result indicates an efficientderacemisation of D/L-glufosinate into L-glufosinate by theRgDAAO/EcgabT enzyme couple.

Example 6: Demonstration of Improved DAAO Enzymes

Using protein mutagenesis strategies as outlined above, improved andvariant DAAO enzymes were identified. The enzymes were assayed accordingto the procedures described below.

Stock Solutions:

The following dye stock solutions were prepared: a 20 mg/mL stocksolution of 2,4,6-tribromo-3-hydroxybenzoic acid (TBHBA) in DMSO; and a100 mg/mL stock solution of 4-aminoantipyrine (4-AAP) in water. Thefollowing enzyme stock solution was prepared: a 1 mg/mL stock solutionof horseradish peroxide (HRP) type 6 in a pH 8.0 potassium phosphatebuffer. The following substrate stock solution was prepared: varyingconcentrations of D or DL amino acid in a pH 8.0 potassium phosphatebuffer.

Reaction Mixes:

The following reaction mixtures were prepared:

Mix A is a combination of the substrate and HRP enzyme. Solutions wereprepared for each substrate concentration to be assayed using reactionbuffer. The solutions were two times the final substrate concentrationand 0.2 mg/mL for the HRP solution.

Mix B is a dye mixture. To 5 mL of reaction buffer was added 120 μL ofTBHBA solution and 400 μL of 4-AAP solution.

Mix C is an enzyme mixture. A 0.1 mg/mL solution of DAAO in reactionbuffer was prepared. The final reaction concentration was 25 μg/mL.

Protocol:

A spectrophotometer was used at a wavelength of 510 nm, whichcorresponds to the maximum absorbance for 4-AAP/TBHBA and is the pointat which the extinction coefficient is 29400 M⁻¹ cm⁻¹. The temperaturefor performing the assays was 30° C. The reaction kinetics were obtainedby measuring every minute for 15 minutes. Between measurements, 20seconds of orbital shaking at normal intensity was performed, followedby 10 seconds of settling time.

Using a 96-well plate, the following mixes (with replicates) were addedin the following order using multi-channel: 100 μl mix A, 50 μl mix B,and 50 μl mix C. The measurements were started immediately after theenzyme addition.

The enzyme kinetics were measured as described above, plotted on aMichaelis Menten graph, and used to calculate Vmax and K_(M). For thevariant Ac302 (54V, 58Q, 213S), the Vmax was 4.2 umol/min*mg.

This analysis was completed for a number of variant DAAO enzymes asabove except that the Mix C stock was 0.2 mg/mL solution of DAAO andthis final reaction concentration of DAAO was 50 ug/mL.

As shown below in Table 1, variant mutant DAAO enzymes showed a range ofactivities:

TABLE 1 Variant Mutations Vmax (% of Ac302) Ac263 54T, 56M, 58K, 213S 33Ac302 54V, 58Q, 213S 100 Ac305 54C, 58H, 213S 88 Ac309 54T, 58T, 213S 71Ac312 54T, 58G, 213S 74 Ac314 54T, 58Q, 213S 99 Ac316 54T, 58S, 213S 75Ac318 54T, 58A, 213S 71 Ac319 54L, 58R, 213S 64 Ac320 54V, 58R, 213S 76Ac322 54V, 58N, 213S 79

Example 7: De-Racemization of Racemic D/L-Glufosinate at a 5 L ReactionSize

The scale of the de-racemization is increased using approaches familiarto those skilled in the art. Reagents and their relative ratios aresubstantially similar to Example 5, but the amounts are significantlygreater. Rather than tubes in shakers, the reactions are performed instirred jacketed reactors, including, optionally, air or oxygen spargingof the broth or headspace. These reactors vary in size, from less than10 mL reaction to tens or hundreds of thousands of liters. Stirringrates are chosen to increase reaction mixing and rate while minimizingpower consumption and shear.

In one example, the reaction was run at the 5 L scale. The system (5 L,30° C.) was run in 200 mM phosphate buffer at a pH of 8.0 in a stirred,jacketed reactor. The following reagents were added essentiallysimultaneously at the start of the reaction: 300 mM D,L-glufosinate, 900mM glutamate, 7.5 g AC302 DAAO, 0.2 g catalase, and 1.0 g E. coli gab Ttransaminase. In addition, 500 mL isopropanol was added to controlfoaming. During the course of the reaction, air was introduced at 0.3VVM (volumes of air per volume of reaction mixture per minute).

HPLC analysis of the reaction demonstrated that equilibrium was reachedwithin 8 hours, with the enantiomeric excess of L-glufosinate overD-glufosinate greater than 99% and the ratio of L-glufosinate to PPO 90%to 10%. This result indicates an efficient deracemisation ofD/L-glufosinate into L-glufosinate by the RgDAAO/EcgabT enzyme couple atthe larger scale.

Example 8: Impact of Oxygen on Reaction Rate

Although stirred, jacketed reactors or immobilized columns typicallyallow for some oxygen transfer, the rate of oxygen uptake afforded bypassive aeration is not sufficient for an efficient process. In oneexample, a reaction was run in the same vessel as Example 7 undersubstantially the same conditions, but under reduced (0.01 VVM), withtwice the AC302 DAAO on a volumetric basis (3 g/L versus 1.5 g/L), andwithout the isopropanol. In this case, the reaction took more than 60hours to achieve equilibrium, demonstrating the critical importance ofaeration for an efficient reaction.

Example 9: Co-Immobilization of DAAO and TA

DAAO and TA enzymes were co-immobilized on EziG controlled pore glassbeads (EnginZyme). 100 mg of EziG type 3 beads were shaken at roomtemperature with 3 ml of solution containing 16 mg of purified AC302DAAO and 1.6 mg of purified gabT in 50 mM potassium phosphate buffer pH7.5, 0.5M NaCl, 20 mM imidazole in a 50 ml Falcon tube. After 30minutes, beads were spun down, immobilizing solution was removed, andbeads were washed 3 times with 10 ml of 100 mM potassium phosphatebuffer pH 7.5.

The reaction was started by adding all other components to the washedbeads. The reaction mix contained 300 mM D/L-glufosinate, 900 mML-glutamic acid, 50 ug catalase, 198 mM potassium phosphate in 2.5 mL.The reaction was incubated at 30 C with shaking (250 rpm) in a 50 mLtube covered in parafilm with holes poked through for gas exchange.

After 1 hour, the depletion of D-glufosinate and formation ofL-glufosinate was determined by HPLC and these rates calculated. After 6hours, beads were spun down, reaction mixture was removed, and beadswere washed 3 times with 10 ml of 100 mM Potassium Phosphate buffer pH7.5. Beads were then stored at 4 C for 18 to 72 hours before thereaction was repeated, for a total of 15 times, after which the retainedactivity was greater than 50% of the initial activity.

Example 10: Effect of Buffer on Reaction

When soluble AC302 DAAO and E. coli gabT TA enzymes are used, phosphatebuffer at >50 mM is required for full activity. A 100 mL reaction wasincubated at 30 C with shaking (250 rpm) in a 500 mL flask covered inparafilm. An air pump was used to bubble air through the reaction forthe first 5 hours. The air pump was removed for overnight incubation sothe reaction would not bubble over and new parafilm with air holes wasused for gas exchange. The reaction mix contained 300 mMD/L-glufosinate, 905 mM L-glutamic acid, 80 mg AC302 DAAO (0.8 mg/mL),14.5 mg gabT (0.145 mg/mL), 2 mg catalase, and isopropanol as anti-foamreagent (10% initial concentration, isopropanol was additionally addedat 2 hr (2 mL), 3 hr (1 mL), 3.5 hr (1 mL), and at 4 hr (2 mL)). 500 uLof 1N NaOH (added before enzymes) was used to adjust pH from about 6 toabout 7. pH remained at about 7 for the entire reaction without furtheradjustment. Due to the potassium phosphate in the stock enzyme buffer,the final mixture was 45 mM phosphate buffer. When compared to a similarreaction with 200 mM phosphate buffer, the reaction rate was 50-60% ofthat of the reaction with the 200 mM buffer.

When immobilized AC302 DOOA and E. coli gatT TA enzymes are used,phosphate buffer of less than 1 mM is sufficient for full activity.Immobilized proteins were prepared and reaction performed as in Example9 for the “Buffered” reaction. In addition, immobilized proteins wereprepared and reactions performed as in Example 9 for the “pH 7”reaction, except that sodium hydroxide was used to adjust the pH of thereaction to pH 7 and no phosphate buffer was added (residual phosphatebuffer from the enzyme storage buffer is less than 1 mM). This workdemonstrated that the initial reaction rate for both the DAAO andcombined DAAO and gabT reactions are very similar with and without theaddition of phosphate buffer when immobilized enzymes are used.

Example 11: Isopropylamine as an Amine Donor

Isopropylamine can be used as an amine donor for conversion of PPO toL-glufosinate with the use of an appropriate TA. PPO was converted toL-glufosinate in a reaction with the following components:

-   -   0.25 mg/mL TA encoded by SEQ ID NO: 1    -   25 mM PPO    -   0.2 mM pyridoxal phosphate    -   250 mM isopropylamine (pHed to 8 w/H3PO4)    -   100 mM Kphos buffer pH 8.0

The reaction was incubated at 25 to 30° C. for 30 hours with gentleshaking (250 rpm). At 0 hours, the amount of L-glufosinate as measuredby HPLC was 0 mM, at 20 hours it was 14 mM, and at 30 hours it was 18mM. This demonstrates that the enzyme encoded by SEQ ID NO: 1 canconvert PPO into L-glufosinate.

Example 12: Lysine as an Amine Donor

Lysine can be used as an amine donor for conversion of PPO toL-glufosinate with the use of an appropriate TA. PPO was converted toL-glufosinate in a reaction with the following components:

-   -   0.4 mg/mL gabT (purified as in Example 3)    -   25 mM PPO (pHed to 8 w/NaOH)    -   0.2 mM pyridoxal phosphate    -   75 mM L-Lysine dihydrochloride (pHed to 8 w/NaOH)    -   100 mM Kphos buffer pH 8.0

The reaction was incubated at 30° C. for 20 hours with shaking (250rpm). L-glufosinate was formed at a rate of 0.4 mM/hr over the 20 hours.This demonstrates that L-lysine can be used to convert PPO toL-glufosinate.

Example 13: Purification and Isolation of L-Glufosinate

Several batches prepared following the procedure described in Example 9but at larger scale were generated. After the beads were removed, eachbatch was heated to 90° C. for at least 10 minutes, and after cooling to20-25° C., filtered to remove a small amount of solids. To eachindividual batch was added 37% HCl, dropwise, to effect theprecipitation of glutamic acid. The amount of 37% HCl added wasapproximately 10% of the volume of the batch. The resulting white solidwas removed by filtration. The batches were combined and concentratedunder vacuum to an oil; the oil contained approximately 153 grams ofL-glufosinate. The oil was diluted with five volumes of water and 37%HCl was added to adjust the solution to pH 1. The solution was treatedsequentially with two portions each of approximately 3.0 kg of prewashedDOWEX 50WX8 cation exchange resin. In each treatment, the solution wasallowed to mix with the resin for 30 minutes after which the resin wasisolated on a filter. Both portions of resins were combined and washedfirst with water and then eluted with 4M NH₄OH. The eluent wasconcentrated under vacuum to an oil; PPO and 2-oxoglutarate were notpresent in the oil. Approximately 100 grams of the oil was diluted withwater and the aqueous ammonium hydroxide was added until the pH wasapproximately 9. To the batch was added 1.0 kg of prewashed DOWEXMonosphere (hydroxide form) anion exchange resin and the mixture wasstirred for approximately 40 minutes. An equal amount of DOWEXMonosphere resin, prewashed, was charged to a glass column. The slurryof DOWEX resin in water was added to the column on top of the prewashedresin. 800 mL of water was charged to the column followed by 0.1 Nacetic acid, which was kept flowing through the column until all of theglutamic acid had eluted as determined by HPLC. 4 N acetic acid was fedto the column until all of the L-glufosinate had eluted from the columnas determined by HPLC. The solution of L-glufosinate was concentratedunder vacuum. The resulting oil was diluted with water and concentratedunder vacuum to minimum volume two times. Methanol was added until aclear solution was obtained and an equal volume of heptane was added.The mixture was concentrated under vacuum to minimum volume and theprocedure was repeated. The remaining 168 grams of oil recovered fromthe cation exchange treatment was treated in a similar fashion to obtainan overall total of 108 grams of crude L-glufosinate. The ratio ofL-glufosinate to glutamic acid was greater than 99:1 as determined byNMR. The resulting solid was mixed with aqueous ammonium hydroxide andconcentrated to dryness to afford 111 grams of L-glufosinate ammonium.Neither methanol nor acetic acid was detected by NMR analysis of theproduct.

It is understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and the terminology is notintended to be limiting. The scope of the invention will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber, which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

All publications, patents, and patent applications cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication, patent, or patent application werespecifically and individually indicated to be incorporated by reference.Furthermore, each cited publication, patent, or patent application isincorporated herein by reference to disclose and describe the subjectmatter in connection with which the publications are cited. The citationof any publication is for its disclosure prior to the filing date andshould not be construed as an admission that the invention describedherein is not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided might be differentfrom the actual publication dates, which may need to be independentlyconfirmed.

It is noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only,” and thelike in connection with the recitation of claim elements, or use of a“negative” limitation. As will be apparent to those of skill in the artupon reading this disclosure, each of the individual embodimentsdescribed and illustrated herein has discrete components and featureswhich may be readily separated from or combined with the features of anyof the other several embodiments without departing from the scope orspirit of the invention. Any recited method may be carried out in theorder of events recited or in any other order that is logicallypossible. Although any methods and materials similar or equivalent tothose described herein may also be used in the practice or testing ofthe invention, representative illustrative methods and materials are nowdescribed.

SEQUENCES SEQ ID NO: 1:MNIAAQSWERREATSFFHTFTDLPSLKTDGPVIIDHGEGPYIIDTVGRRYFEGNSGLWNMTLGFSERRLSDAALKQYQEFPGYHTFFGRNSKPTVELAERMLKLAPAPMSRVFFTNSGSEANESIVKLLWMMWAAEGRPERRKLLTRKNAYHGATVMASALTGKDYVKAFGLPGPEIVTLDCPHAWRFALPGEGDDEFAARLAANLETRILQEGPETIAGMFAEPVMGAGGVIVPPATYFAKIQPVLQRYGIPLIADEVICGFGRTGSLWGTLAVGQQPDIIVASKSMSAGYFPMGAVMLSADIDKRATAASEVWEEFPHGFTTGGHPVGCAISLEAIRIITEEGVFENVKSVSETFQSGLRALADHPMIGEARGMGLMGALETVADKKTKQSFSGDLRIGERISKEARDRGFIIRPLGSSVVLAPPFISTHGQIEELLAV LKEVLDVVYGTVKGEVASEQ ID NO: 2: MHSQKRVVVLGSGVIGLSSALILARKGYSVHILARDLPEDVSSQTFASPWAGANWTPFMTLTDGPRQAKWEESTFKKWVELVPTGHAMWLKGTRRFAQNEDGLLGHWYKDITPNYRPLPSSECPPGAIGVTYDTLSVHAPKYCQYLARELQKLGATFERRTVTSLEQAFDGADLVVNATGLGAKSIAGIDDQAAEPIRGQTVLVKSPCKRCTMDSSDPASPAYIIPRPGGEVICGGTYGVGDWDLSVNPETVQRILKHCLRLDPTISSDGTIEGIEVLRHNVGLRPARRGGPRVEAERIVLPLDRTKSPLSLGRGSARAAKEKEVTLVHAYGFSSAGYQQSWGAAEDVAQLVDEAFQRYHGAARESKL SEQ ID NO: 3:MNSNKELMQRRSQAIPRGVGQIHPIFADRAENCRVWDVEGREYLDFAGGIAVLNTGHLHPKVVAAVEAQLKKLSHTCFQVLAYEPYLELCEIMNQKVPGDFAKKTLLVTTGSEAVENAVKIARAATKRSGTIAFSGAYHGRTHYTLALTGKVNPYSAGMGLMPGHVYRALYPCPLHGISEDDAIASIHRIFKNDAAPEDIAAIVIEPVQGEGGFYASSPAFMQRLRALCDEHGIMLIADEVQSGAGRTGTLFAMEQMGVAPDLTTFAKSIAGGFPLAGVTGRAEVMDAVAPGGLGGTYAGNPIACVAALEVLKVFEQENLLQKANDLGQKLKDGLLAIAEKHPEIGDVRGLGAMIAIELFEDGDHNKPDAKLTAEIVARARDKGLILLSCGPYYNVLRILVPLTIEDAQIRQGLEIISQCFDEAKQ

What is claimed is:
 1. A herbicidal formulation, wherein saidformulation comprises L-glufosinate at an enantiomeric excess of greaterthan 90% over D-glufosinate, and more than 0.01% but less than 10% PPO(2-oxo-4-(hydroxy(methyl)phosphinoyl)butyric acid), said formulationfurther comprising at least one adjuvant.
 2. The formulation of claim 1,wherein said adjuvant is selected from a surfactant, a solvent, apolysaccharide humectant, and a diluent.
 3. The formulation of claim 2,wherein said surfactant is sodium alkyl ether sulfate.
 4. Theformulation of claim 2, wherein said solvent is selected from at leastone of 1-methoxy-2-propanol, dipropylene glycol, and ethylene glycol. 5.The formulation of claim 2, wherein said polysaccharide humectant isselected from at least one of alkyl polysaccharides, pentoses, highfructose corn syrup, sorbitol, and molasses.
 6. The formulation of claim2, wherein said diluent is an aqueous component.
 7. The formulation ofclaim 2, wherein said diluent is water.
 8. The formulation of claim 1,wherein said L-glufosinate composition comprises L-glufosinate ammoniumin an amount from 10-30% by weight of the formulation; and wherein saidadjuvant comprises one or more additional components selected from thegroup consisting of sodium alkyl ether sulfate in an amount from 10-40%by weight of the formulation; 1-methoxy-2-propanol in an amount from0.5-2% by weight of the formulation; dipropylene glycol in an amountfrom 4-18% by weight of the formulation; and alkyl polysaccharide in anamount from 4-20% by weight of the formulation; and water as the balanceof the formulation.
 9. The formulation of claim 1, wherein theL-glufosinate composition comprises L-glufosinate ammonium in an amountof 12.25% by weight of the formulation; and wherein said adjuvantcomprises sodium alkyl ether sulfate in an amount of 31.6% by weight ofthe formulation; 1-methoxy-2-propanol in an amount of 1% by weight ofthe formulation; dipropylene glycol in an amount of 8.6% by weight ofthe formulation; alkyl polysaccharide in an amount of 9.8% by weight ofthe formulation; and water in an amount of 36.75% by weight of theformulation.
 10. The formulation of claim 1, wherein the L-glufosinatecomposition comprises L-glufosinate ammonium in an amount of 24.5% byweight of the formulation; and wherein said adjuvant comprises sodiumalkyl ether sulfate in an amount of 31.6% by weight of the formulation;1-methoxy-2-propanol in an amount of 1% by weight of the formulation;dipropylene glycol in an amount of 8.6% by weight of the formulation;alkyl polysaccharide in an amount of 9.8% by weight of the formulation;and water in an amount of 24.5% by weight of the formulation.
 11. Theformulation of claim 1, wherein the L-glufosinate composition comprisesL-glufosinate ammonium in an amount of 12.25% by weight of theformulation; sodium alkyl ether sulfate in an amount of 15.8% by weightof the formulation; 1-methoxy-2-propanol in an amount of 0.5% by weightof the formulation; dipropylene glycol in an amount of 4.3% by weight ofthe formulation; alkyl polysaccharide in an amount of 4.9% by weight ofthe formulation; and water in an amount of 62.25% by weight of theformulation.
 12. The formulation of claim 1, wherein the L-glufosinatecomposition comprises L-glufosinate ammonium in an amount from 10-30% byweight of the formulation; and wherein said adjuvant is selected fromone or more additional components selected from the group consisting ofsodium alkyl ether sulfate in an amount from 10-40% by weight of theformulation; 1-methoxy-2-propanol in an amount from 0.5-2% by weight ofthe formulation; and alkyl polysaccharide in an amount from 3-10% byweight of the formulation; and water as the balance of the formulation.13. The formulation of claim 1, wherein the L-glufosinate compositioncomprises L-glufosinate ammonium in an amount of 12.25% by weight of theformulation; and wherein said adjuvant comprises sodium alkyl ethersulfate in an amount of 22.1% by weight of the formulation;1-methoxy-2-propanol in an amount of 1% by weight of the formulation;alkyl polysaccharide in an amount of 6.2% by weight of the formulation;and water in an amount of 58.45% by weight of the formulation.
 14. Theformulation of claim 1, wherein the L-glufosinate composition comprisesL-glufosinate ammonium in an amount of 24.5% by weight of theformulation; and wherein said adjuvant comprises sodium alkyl ethersulfate in an amount of 22.1% by weight of the formulation;1-methoxy-2-propanol in an amount of 1% by weight of the formulation;alkyl polysaccharide in an amount of 6.2% by weight of the formulation;and water in an amount of 46.2% by weight of the formulation.
 15. Theformulation of claim 1, wherein the L-glufosinate composition comprisesL-glufosinate ammonium in an amount of 12.25% by weight of theformulation; and wherein said adjuvant comprises sodium alkyl ethersulfate in an amount of 11.05% by weight of the formulation;1-methoxy-2-propanol in an amount of 0.5% by weight of the formulation;alkyl polysaccharide in an amount of 3.1% by weight of the formulation;and water in an amount of 73.1% by weight of the formulation.
 16. Amethod for selectively controlling weeds in an area comprising applyingan effective amount of the formulation of claim 1 to the area.
 17. Themethod of claim 16, wherein said area comprises a crop of planted seedsor crops that are resistant to glufosinate.